Methods and agents for treating alzheimer&#39;s disease

ABSTRACT

The present disclosure provides compositions and methods useful for treating or preventing diseases or disorders where beta amyloid accumulation or aggregation contributes to the pathology or symptomology of the disease, for example Alzheimer&#39;s disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of the U.S.Provisional Application No. 61/805,735, filed Mar. 27, 2013, the contentof which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates generally to methods and compositions fordecreasing, inhibiting or reducing beta amyloid accumulation. Thepresent disclosure also provides methods and compositions for treatmentand/or prevention of neurodegenerative diseases and disorders.

BACKGROUND

Alzheimer's disease (AD) is the most prevalent neurodegenerative diseaseand the leading cause of dementia among the elderly. The mechanismsunderlying the onset and progression of neurodegeneration and cognitivedecline are incompletely understood. A major breakthrough in ourunderstanding of AD was the identification of gene mutations associatedwith rare familial AD (FAD) cases. Autosomal dominant mutations in theamyloid beta (A4) precursor protein (APP) and presenilin 1 and 2(PSEN1/2) genes greatly accelerate the rate of cognitive decline leadingto early-onset dementia (Bertram et al., 2010; Tanzi, 2012). The vastmajority of AD cases, however, are late-onset forms (LOAD), which lackan obvious Mendelian inheritance pattern. LOAD has a strong geneticcomponent and is likely caused by a combination of multiple riskalleles, each with modest and partially penetrant effects, andenvironmental factors (Bertram et al., 2010). Although apolipoprotein Eε4 (APOE ε4) remained for a long time the only confirmed genetic riskfactor for LOAD, it accounts for only 10-20% of the LOAD risk,suggesting the existence of additional risk factors (Liu et al., 2013).Recently, genome-wide association studies (GWAS) performed on extendedcohorts (thousands of individuals) led to the identification ofadditional confirmed genetic risk factors for AD: CD33 (Bertram et al.,2008; Hollingworth et al., 2011; Naj et al., 2011), CLU, BIN1, PICALM,CR1, CD2AP, EPHA1, ABCA7, MS4A4A/MS4A6E (Harold et al., 2009;Hollingworth et al., 2011; Lambert et al., 2009; Naj et al., 2011;Seshadri et al., 2010) and TREM2 (Guerreiro et al., 2013; Jonsson etal., 2013). Understanding the molecular and cellular activities of thesenovel genes, as well as their functional interactions, should greatlyadvance our understanding of AD.

The deposition of amyloid beta (Aβ)-containing plaques is a pathologicalhallmark of both FAD and LOAD. Aβ results from the amyloidogenicprocessing of APP, which is cleaved by the sequential action ofβ-secretase/BACE1 and γ-secretase/Presenilin (Querfurth and LaFerla,2010). In FAD, both APP and PSEN1/2 mutations lead to enhancedamyloidogenic processing of APP and enhanced production of the toxicAβ42 species (Querfurth and LaFerla, 2010). Less is known about themechanisms of Aβ formation, self-assembly and clearance in LOAD.Interestingly, several genes linked to LOAD have been shown to impact Aβgeneration, aggregation, or clearance (Bertram et al., 2010), suggestingthat Aβ dysregulation is a central pathogenic mechanism in LOAD. Awidely accepted model of AD pathogenesis is the “amyloid hypothesis”whereby increased production and self-assembly of Aβ toxic speciesinitiates a series of progressive changes that ultimately lead toneurodegeneration (Hardy and Selkoe, 2002; Hardy and Higgins, 1992;Tanzi and Bertram, 2005). In this hypothesis, persistent Aβ proteotoxicstress triggers the hyperphosphorylation and aggregation of themicrotubule associated protein tau leading to neurofibrillary tangles,another pathological hallmark of AD (Tanzi and Bertram, 2005).Therefore, a better understanding of the mechanisms that regulate thegeneration and deposition, as well as clearance, of Aβ might improve thetherapeutic approaches in AD.

Two single nucleotide polymorphisms (SNPs) in the CD33, rs3826656(Bertram et al., 2008) and rs3865444 (Hollingworth et al., 2011; Naj etal., 2011), have been associated with LOAD. The 67 kDa type 1transmembrane protein CD33 (Siglec-3) is a member of the sialicacid-binding immunoglobulin-like lectins (Siglecs) and is expressed inimmune and hematopoietic cells. The Siglecs recognize sialic acidresidues of glycoproteins and glycolipids, have one or moreimmunoreceptor tyrosine-based inhibition motifs (ITIMs) and mediatecell-cell interactions that inhibit or restrict immune responses(Crocker et al., 2012; Pillai et al., 2012). CD33 activity has beenimplicated in several processes such as: adhesion processes in immune ormalignant cells, endocytosis, inhibition of cytokine release bymonocytes and immune cell growth (Crocker et al., 2007; von Gunten andBochner, 2008). To date, no functions have been described for CD33 inthe brain.

SUMMARY

The present disclosure is based on inventors' discovery that CD33protein inhibits uptake and clearance of beta amyloid in microglialcells. Inventors have shown that brain levels of insoluble Aβ-42 as wellas amyloid plaque burden are markedly reduced in CD33^(−/−) mice.Inventors have also demonstrated that CD33 inactivation mitigates Aβpathology. Accordingly, in one aspect, the present disclosure relatesgenerally to methods decreasing beta amyloid (“Aβ”) accumulation byinhibition of CD33, for example inhibition of expression and/or activityof CD33 protein. The methods disclosed herein can be used fordecreasing, inhibiting or reducing beta amyloid accumulation in asubject, for example in brain of the subject, in need thereof. In someembodiments, the beta amyloid is Aβ-42.

In some embodiments, the methods as disclosed herein compriseadministering to a subject in need of decreasing, inhibiting or reducingbeta amyloid accumulation, an effective amount of an agent that inhibitsor reduces the expression or activity of CD33 protein.

In some other embodiments, the methods as disclosed herein compriseadministering to a subject in need of decreasing, inhibiting or reducingbeta amyloid accumulation, a CD33 protein or polypeptide that lackssialic acid binding domain or a nucleic acid encoding such CD33 protein.A CD33 protein lacking the sialic acid binding domain is also referredto as a non-sialic CD33 protein or polypeptide. In some embodiments, thenucleic acid encoding the non-sialic CD33 protein is a modified RNA. Insome embodiments, the nucleic acid encoding the non-sialic CD33 proteinis a vector, e.g., an expression vector.

Without wishing to be bound by a theory, decreasing, inhibiting orreducing beta amyloid accumulation in a subject can be useful fortreatment and/or prevention of diseases or disorders where beta amyloidaccumulation or aggregation contributes to the pathology or symptomologyof the disease. Thus, the disclosure also provides methods or treatmentand/or prevention of diseases or disorders where beta amyloidaccumulation or aggregation contributes to the pathology or symptomologyof the disease. In some embodiments, the disclosure also providesmethods or treatment and/or prevention of neuro-inflammatory diseasesand disorders by inhibition CD33, for example inhibition of expressionand/or activity of CD33 protein. It is not intended that the presentinvention to be limited to any particular stage of the disease (e.g.early or advanced).

In some embodiments, the methods as disclosed herein compriseadministering to a subject in need of treatment and/or prevention of adisease or disorder where beta amyloid accumulation or aggregationcontributes to the pathology or symptomology of the disease (e.g., aneuro-inflammatory disease), an effective amount of an agent thatinhibits or reduces the expression or activity of CD33 protein.

In some embodiments, the methods as disclosed herein compriseadministering to a subject in need of treatment and/or prevention of adisease or disorder where beta amyloid accumulation or aggregationcontributes to the pathology or symptomology of the disease (e.g., aneuro-inflammatory disease), a CD33 protein that lacks sialic acidbinding domain or a nucleic acid encoding such a CD33 protein.

In some embodiments, where beta amyloid accumulation or aggregationcontributes to the pathology or symptomology of the disease results in adecline in cognitive function, for example Alzheimer's Disease, themethod of treatment and/or prevention of the disease using the methodsas disclosed herein can further comprise assessing the cognitivefunction of the subject after administration. In some embodiments, themethod can comprise assessing presence of Aβ-42, for example, in theCSF.

In some embodiments, the methods disclosed herein can further compriseadministering to the subject additional therapeutic agents, for examplebut not limited to therapeutic agents used in the treatment ofneurodegenerative disorders. For example, where the neurodegenerativedisorder is, for example, Alzheimer's Disease, the subject can befurther administered therapeutic agents for the treatment of Alzheimer'sDisease, for example but are not limited to ARICEPT or donepezil, COGNEXor tacrine, EXELON or rivastigmine, REMINYL or galantamine, anti-amyloidvaccine, Aβ-lowering therapies, mental exercise or stimulation.

In some embodiments, the methods as disclosed herein are applicable tosubjects, for example mammalian subjects. In some embodiments, thesubject is a human.

Without limitation, the agents that inhibits the expression or activityof the CD33 protein can be selected from the group consisting of smallor large organic or inorganic molecules, nucleic acids, nucleic acidanalogs and derivatives, peptides, peptidomimetics, proteins, antibodiesand antigen binding fragments thereof, monosaccharides, disaccharides,trisaccharides, oligosaccharides, polysaccharides, lipids,glycosaminoglycans, an extract made from biological materials, and anycombinations thereof.

In some embodiments, the agent is a nucleic acid. A nucleic acid agentcan be selected from, for example, siRNA, shRNA, miRNA, anti-microRNA,antisense RNA or oligonucleotide, aptamer, ribozyme, and anycombinations thereof. When the agent is a nucleic acid, the agent itselfcan be administered to the subject or a vector expressing/encoding theagent can be administered to the subject. In some embodiments, thevector expressing/encoding the agent is an Adeno-associated virus (AAV)vector.

In some embodiment, the agent is a small molecule, for example, but notlimited to, a small molecule reversible or irreversible inhibitor ofCD33 protein.

In some embodiments, the agent is an antibody. Antibody can be apolyclonal or monoclonal antibody. Further, the antibody can be achimeric antibody. In some embodiments, the antibody is a humanizedantibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show increased CD33 expression in AD. (FIG. 1A) QuantitativeRT-PCR analysis of CD33 expression in the frontal cortex revealsincreased CD33 mRNA levels in AD relative to control subjects; CD33 mRNAlevels have been normalized to GAPDH or β-Actin mRNA. (FIG. 1B) Westernblotting detection of CD33 in the frontal cortex reveals a markedupregulation in AD cases relative to age-matched controls. A lesspronounced increased expression is also seen for Iba1. GAPDH served asloading control. (FIG. 1C) The CD33/GAPDH and CD33/Iba1 protein ratiosare increased in AD patients relative to age-matched controls. (FIG. 1D)The levels of CD33 protein are decreased in carriers of the protectiveminor (T) allele of the CD33 SNP rs3865444. Bar graph showing CD33protein levels in individuals from the indicated groups (CTRL or AD) andgenotypes (G/G versus G/T or T/T). For (FIGS. 1A-1D): *p<0.05, **p<0.01and ***p<0.001, student's t-test. Data are represented as mean±SEM.(FIG. 1E) Analysis based on general linear regression model reveals thatthe protective minor (T) allele of rs3865444 is associated withdecreased CD33 protein, but not mRNA levels in both controls and ADcases (p<0.05 was considered statistically significant). See also FIG. 8and Table 1.

FIGS. 2A-2M show increased number of CD33-immunoreactive microglia inAD. (FIGS. 2A-2A″, FIGS. 2C-2C″) Fluorescent immunolabeling revealsco-localization between CD33 (visualized as red) and the microglialmarker Iba1 (visualized as green) in the frontal cortex of CTRL (FIGS.2A-2A″) and AD (FIGS. 2C-2C″) subjects. (FIGS. 2B and 2D) CD33 labelingusing diaminobenzidine reveals numerous microglial cells that arepositive for CD33. (FIG. 2E) Stereology-based quantification revealsincreased numbers of CD33-positive microglial cells in the frontalcortex of AD cases (n=28) relative to controls (n=18), ***p<0.001,student's t-test. (FIG. 2F) CD33 protein levels normalized to Iba1protein levels positively correlate with the numbers ofCD33-immunoreactive microglia in the AD cases (n=25; r=0.525; p=0.007,Pearson's correlation test). (FIGS. 2G, 2H) CD33 protein levelscorrelate with the levels of the microglial marker Iba1, both incontrols (n=15; r=0.543; p=0.03, Pearson's correlation test) and ADcases (n=25; r=0.582; p=0.002, Pearson's correlation test). (FIGS.2I-2K) Carriers of the protective minor (T) allele of rs386544 exhibitdecreased numbers of CD33-positive microglia. Shown are representativepictures from the frontal cortex of a (FIG. 2I) G/G carrier, (FIG. 2J)G/T carrier and (FIG. 2K) T/T AD carrier, stained for CD33. (FIG. 2L)Stereology-based quantifications reveal reduced numbers of CD33-positivemicroglia in carriers of two protective (T) alleles of rs3865444(**p<0.01 T/T carriers versus G/G carriers, one-way Kruskal-WallisANOVA, Dunn's test). Data are represented as mean±SEM. Scale barsrepresent 25 μm. (FIG. 2M) The protective (T) allele of rs3865444 isassociated with decreased numbers of CD33-immunoreactive microglia inboth controls and AD cases (general linear regression model, p<0.05 wasconsidered statistically significant). See also FIG. 9.

FIGS. 3A-3D show that CD33 microglial expression positively correlateswith formic acid-soluble Aβ42 levels and amyloid plaque burden in AD.(FIG. 3A) Formic acid (FA)-soluble Aβ42 levels are decreased in carriersof the rs3865444 minor (T) allele. ELISA analysis of Aβ40 and Aβ42 inFA-soluble fractions isolated from the frontal cortex of AD cases of theindicated genotypes (**p<0.01, student's t-test). Data are representedas mean±SEM. (FIG. 3B) The protective rs3865444 (T) allele is associatedwith decreased levels of both FA-soluble Aβ42 and TBS-soluble Aβ40 in ADcases (n=25 AD cases, n=15 controls, general linear regression model,p<0.05 was considered statistically significant). (FIG. 3C) The numbersof CD33-immunoreactive microglia positively correlate with theFA-soluble Aβ42 levels in AD cases (n=25; r=0.446; p=0.02, Spearman'scorrelation test). (FIG. 3D) The numbers of CD33-positive microgliapositively correlate with amyloid plaque burden in AD brain (n=25;r=0.471; p=0.017; Spearman's correlation test). See also FIG. 10.

FIGS. 4A-4E show that CD33 inactivation leads to increased microglialuptake of Aβ42. (FIGS. 4A-4A′, FIGS. 4B-4B′″) Primary microglial cellcultures were incubated with fluorescently labeled Aβ42 for 3 hours andthen subjected to immunofluorescent labeling for CD33 (FIG. 4A, FIG. 4B)and Iba1 (FIG. 4A′, FIG. 4B′″). Microglial cultures derived fromCD33^(−/−) mice, at postnatal day 1, exhibited a markedly increased Aβ42uptake (compare FIG. 4A′ and FIG. 4B′). Scale bar represents 25 μM.(FIG. 4C) Quantification of Aβ42 average signal intensity in individualcells reveals a strong increase in Aβ42 signal in CD33^(−/−) cellsrelative to WT cells (at least 30 cells were scored per genotype,**p<0.01, student's t-test). (FIGS. 4D, 4E) Microglial cell cultureswere treated with unlabeled Aβ42 and incubated for 3 hours. Afterincubation, cells were either collected for ELISA analysis (t=0) orwashed and incubated for an additional 3 hours in Aβ-free medium,followed by ELISA analysis (t=3 hrs). (FIG. 4D) CD33−/− microgliaexhibit increased Aβ42 uptake levels relative to WT microglia (t=0)(results were derived from 4 independent experiments; ***p<0.001,student's t-test). (FIG. 4E) Similar rates of Aβ42 degradation inCD33^(−/−) and WT microglial cells incubated in the absence of Aβ42 foran additional 3 hours (the percentage of remaining Aβ42 was the ratio ofAβ42 remaining at t=3 hours to the total amount of Aβ42 internalized att=0). Data are represented as mean±SEM. See also FIG. 11.

FIGS. 5A-5G shows that CD33 inhibits microglial uptake of Aβ42. (FIGS.5A-D′″) BV2 microglial cells were incubated with fluorescently labeledAβ42 for 3 hours and then subjected to immunofluorescent labeling forCD33 (FIGS. 5A-5D) and Iba1 (FIGS. 5A′″-5D′″). Overexpression of WT-CD33decreases the amount of internalized Aβ42 (compare FIG. 5A′ and FIG.5B′). Expression of a ubiquitylation-defective CD33 variant (K7R-CD33)further decreases the amount of internalized Aβ42 (compare FIG. 5B′ andFIG. 5C′). A CD33 variant lacking the sialic acid-binding V-typeimmunoglobulin-like extracellular domain (ΔV-Ig-CD33) no longer inhibitsAβ42 uptake (compare FIG. 5B′, FIG. 5C′ with FIG. 5D′). Scale barrepresents 25 μM. (FIG. 5E) Quantification of Aβ42 signal intensity inindividual cells transfected with the indicated constructs or with anempty vector; at least 30 cells were scored per condition (*p<0.05,**p<0.01, ***p<0.001, one-way ANOVA, Tukey's test). The series from leftto right represents empty vector, GFP, CD33^(WT), CD33^(K7R),CD33^(ΔV-Ig). (FIG. 5F, FIG. 5G) BV2 cells were treated with unlabeledAβ42 and incubated for 3 hours. The series from left to right representsempty vector, GFP, CD33^(WT), CD33^(K7R), cD33^(ΔV-Ig). Afterincubation, cells were either collected for ELISA analysis (t=0) orwashed and incubated for an additional 3 hours in Aβ-free medium,followed by ELISA analysis (t=3 hours). (FIG. 5F) WT-CD33 inhibitsmicroglial uptake of Aβ42. ELISA quantifications of Aβ42 levels in BV2cells transfected with the indicated constructs or with empty vector(results were obtained from 4 independent experiments; *p<0.05,**p<0.01, one-way ANOVA, Tukey's test). (FIG. 5G) CD33 overexpressiondoes not affect the rate of Aβ42 degradation by BV2 cells (thepercentage of remaining Aβ42 was the ratio of Aβ42 remaining at t=3hours to the total amount of Aβ42 internalized at t=0). Both emptyvector and a GFP-expressing vector served as controls. Data arerepresented as mean±SEM.

FIGS. 6A-6F shows that CD33 deletion decreases formic acid-soluble Aβ42levels in APP/PS1 mice. (FIG. 6A) Western blotting analysis of corticalextracts reveals increased levels of APP and APP C-terminal fragments(CTFs) in four month-old APP/PS1 mice in comparison to controls.However, APP and APP-CTFs levels are similar in APP/PS1 andAPP/PS1/CD33^(−/−) mice. β-Actin served as loading control. (FIG. 6B)Quantification of APP, α-CTF and β-CTF levels in APP/PS1 andAPP/PS1/CD33−/− mice (n=9 male mice were analyzed per group). (FIGS.6C-6F) ELISA analysis of Aβ40 (FIG. 6C, FIG. 6E) and Aβ42 (FIG. 6D, FIG.6F) in TBS-soluble (FIG. 6C, FIG. 6D) or formic acid (FA)-soluble (FIG.6E, FIG. 6F) fractions isolated from the cortex of four month-old malemice of the indicated genotypes. APP/PS1 mice exhibit increased levelsof Aβ40 and Aβ42 relative to WT and CD33^(−/−) mice, as expected. Nodifferences in Aβ40 levels and TBS-soluble Aβ42 levels were seen inAPP/PS1 and APP/PS1/CD33^(−/−) mice. However, the FA-soluble Aβ42 levelswere markedly decreased in APP/PS1/CD33^(−/−) relative to APP/PS1 mice(n=9-12 mice were analyzed per group, *p<0.05, one-way Kruskal-WallisANOVA, Dunn's test). Data are represented as mean±SEM. For FIGS. 6C-6F,the series from left to right represents WT, CD33^(−/−), APP/PS1,APP/PS1/CD33^(−/−), APP/PS1/CD33^(−/−). See also FIG. 12.

FIGS. 7A-7F shows that CD33 deletion mitigates Aβ plaque pathology inAPP/PS1 mice. (FIGS. 7A-7D) Photomicrographs of selected cortical (FIG.7A, FIG. 7B) and hippocampal (FIG. 7C, FIG. 7D) fields from 6-7month-old APP/PS1 (FIG. 7A, FIG. 7C) and APP/PS1/CD33^(−/−) brains (FIG.7D, FIG. 7E) were stained with the anti-Aβ antibody 3D6 to reveal Aβplaques. The Aβ plaque burden is decreased in APP/PS1/CD33^(−/−) brainsrelative to APP/PS1 brains (compare FIG. 7B with FIG. 7A and FIG. 7Dwith FIG. 7C). (FIG. 7E, FIG. 7F) Quantification of amyloid plaqueburden in the cortex (FIG. 7E) and hippocampus (FIG. 7F) of 6-7month-old APP/PS1 and APP/PS1/CD33−/− brains (n=9-11 male mice wereanalyzed per group, *p<0.05, **p<0.01, student's t-test). Data arerepresented as mean±SEM. Scale bar is 50 μm.

FIGS. 8A-8C show decreased levels of CD33 protein in carriers of thers3865444 minor (T) allele. (FIG. 8A) Schematic diagram of thetranscribed CD33 RNA (CD33 pre-mRNA; upper) and CD33 mRNA (lower). Thesets of primers (Forward F1 and reverse R1 targeting exons 3-4; forwardF2 and reverse R2 targeting exons 4-5) used for quantitative RT-PCR areshown by the arrows. (FIG. 8B) Results of qRT-PCR using the indicatedsets of primers. mRNA was isolated from frozen cortical extractsobtained from AD cases (n=25) and age-matched controls (n=15) of theindicated genotypes. Each series represents CTRL G/G, CTRL G/T+T/T, ADG/G, AD G/T+T/T from left to right. (FIG. 8C) CD33 protein levels aredecreased in carriers of the rs3865444 minor (T) allele. Westernblotting was used to quantify CD33 protein levels in AD cases (n=25) andage-matched controls (n=15) of the indicated genotypes. CD33 expressionwas normalized to GAPDH or to the microglial marker Iba1 (*p<0.05,student's t-test). Data are represented as mean±SEM. Each seriesrepresents CTRL G/G, CTRL G/T, CTRL T/T, AD G/G, AD G/T, AD T/T fromleft to right.

FIGS. 9A-9I″ show CD33 expression pattern in the human brain. (FIG. 9A)Stereology-based quantifications of the number of CD33-positive cells inthe frontal cortex of AD cases (n=28) and age-matched controls (n=18)(***p<0.001, student's t-test). (FIG. 9B) Stereology-basedquantifications of the number of CD33-positive neurons in the frontalcortex of AD cases (n=28) and age-matched controls (n=18) (p=n.s.,Mann-Whitney U test). (FIG. 9C) Distribution of CD33-positive cells inAD cases and age-matched controls. Most CD33 cells are microglial, andonly a minor fraction corresponds to neurons. (FIGS. 9D-9D″, FIGS.9E-9E″) Fluorescent immunolabeling reveals co-localization between CD33(visualized as red) and the neuronal marker MAP2 (visualized as green)in controls (FIGS. 9D-9D″) as well as AD cases (FIGS. 9E-9E″). (FIG. 9F)Quantification of CD33 levels within MAP2-positive neurons (*p<0.05,student's t-test). Data are represented as mean±SEM. (FIGS. 9G-9I) CD33is not expressed in astrocytes, endothelial cells or oligodendrocytes inthe aged human brain. Immunolabeling for CD33 (visualized as red; FIGS.9G-9I) and astrocytic (GFAP; visualized as green; FIG. 9G′), endothelial(von Willebrand factor [VWF]; visualized as green; FIG. 9H′), oroligodendrocytic (myelin basic protein [MBP]; visualized as green; FIG.9I′) markers reveals no co-localization between CD33 signal and thesemarkers. Scale bar is 25 μm.

FIGS. 10A-10C′″ show localization of CD33 and relationship to amyloidplaques. (FIGS. 10A-10C) Frontal cortices from AD cases wereimmunolabeled for CD33 (visualized as red; FIGS. 10A′, 10B′, and 10C′),the microglial marker Iba1 (visualized as white; FIGS. 10A′, 10B′″, and10C′″) and stained with Thioflavin S (visualized as green; FIGS. 10A,10B, and 10C) to detect amyloid plaques. CD33 exhibited a prominentmicroglial localization that was broadly distributed throughout thefrontal cortex. In addition, an increased density of CD33-positivemicroglia was noted around amyloid plaques (white circles; FIG. 10A″,FIG. 10B″). (FIGS. 10C-10C′″) Higher-magnification views of the selectedAβ plaque (white arrow in FIG. 10B″). Scale bar is 25 μm in FIG. 10A′″and FIG. 10B′″ and 5 μm in FIG. 10C′″.

FIGS. 11A-11C shows mCD33-specific polyclonal antibody. (FIG. 11A) Shownis a schematic representation of the mouse CD33 (mCD33) protein,highlighting the location of the immunizing peptide (shaded area in thefirst line; residues 18-32). The amino acid sequence is SEQ ID NO 1.(FIG. 11B) The rabbit polyclonal antibody raised against this CD33peptide was tested on protein extracts derived from HEK293 cellstransfected with either an empty vector or a vector encoding full-lengthmouse CD33. GAPDH served as loading control. (FIG. 11C) Western blottingusing the anti-CD33 rabbit polyclonal antibody and cortical lysatesreveals CD33 expression in WT mice and absence of CD33 expression inCD33^(−/−) mice. GAPDH served as loading control.

FIGS. 12A-12U show that APP/PS1/CD33^(−/−) mice do not display enhancedmicroglial recruitment or accelerated astrogliosis in comparison toAPP/PS1 mice. Shown are representative microphotographs of cortical(FIGS. 12A-12D, FIGS. 12L-12O) and hippocampal (FIGS. 12E-12H, FIGS.12P-12S) coronal sections stained for Iba1 (FIGS. 12A-12H) and GFAP(FIGS. 12L-12S). (FIG. 12I) Quantification of Iba1-positive microglialcell numbers reveals similar microglia numbers in the hippocampus andcortex in mice of the indicated genotypes (n=5-6 male mice/genotype, 4month-old). (FIG. 12J) Western blotting using a Iba1 antibody alsoreveals no changes in Iba1 protein levels in the cortex of mice of theindicated genotypes. (FIG. 12K) Quantifications of Iba1 protein levels,using normalization to β-Actin (n=6 male mice/genotype, 4 month-old).(FIG. 12T) Quantification of GFAP-positive astrocyte numbers revealssimilar astrogliosis in the cortex of WT and CD33^(−/−) mice and moreastrogliosis in APP/PS1 and APP/PS1/CD33^(−/−) mice; however, the extentof astrogliosis was similar in the cortex of APP/PS1 andAPP/PS1/CD33^(−/−) mice (n=5-6 male mice/genotype, 4 month-old). (FIG.12U) Quantification of the GFAP-immunoreactive cell numbers in thehippocampus reveals a similar degree of astrogliosis in the hippocampusin all groups (n=5-6 male mice/genotype, one-way Kruskal-Wallis ANOVA,Dunn's test). Data are represented as mean±SEM. Scale bar is 25 μm.

DETAILED DESCRIPTION

In part, this invention is based on the inventors' discovery that CD33protein inhibits uptake and clearance of beta amyloid in microglialcells and brain levels of insoluble Aβ-42 as well as amyloid plaqueburden are markedly reduced in CD33^(−/−) mice. In particular,decreasing, inhibiting, or reducing the expression or activity of CD33protein led to reduction of amyloid, for example beta-amyloid (or Aβ-42)accumulation. As the work disclosed herein shows, CD33 inactivation canmitigate Aβ pathology.

Therefore, the inventors have discovered that inactivation of CD33,e.g., with CD33, inhibitors can be used in the treatment and/orprevention of neuro-inflammatory disease and disorders. As used herein,the term “neuro-inflammatory disease” refers to an inflammatory diseaseor disorder in the central nervous system (CNS, brain, and spinal cord),and includes, but is not limited to, neurodegenerative diseases anddisorders.

CD33 is a transmembrane, immunoglobulin-like lectin that normally has asialic acid-binding domain. CD33 is also known as SIGLEC3, and itencodes a cell-surface receptor on cells of monocytic or myeloidlineage, and regulates the innate immune system (Bertram et al., Am. J.Hum. Gnet. 2008, 83, 623-632). The amino acid sequence for CD33 is knownin the art and is provided below for reference: mplllllpll wagalamdpnfwlqvqesvt vqeglcvlvp ctffhpipyy dknspvhgyw fregaiisrd spvatnkldqevqeetqgrf rllgdpsrnn cslsivdarr rdngsyffrm ergstkysyk spqlsvhvtdlthrpkilip gtlepghskn ltcsyswace qgtppifswl saaptslgpr tthssvliitprpqdhgtnl tcqvkfagag vttertiqln vtyvpqnptt gifpgdgsgk qetragvvhgaiggagvtal lalcicliff ivkthrrkaa rtavgrndth pttgsaspkh gkksklhgptetsscsgaap tvemdeelhy aslnfhgmnp skdtsteyse vrtq (SEQ ID NO: 2)

As used herein, the term “neurodegenerative disease” refers to a variedassortment of central nervous system disorders characterized by gradualand progressive loss of neural tissue and/or neural tissue function. Aneurodegenerative disease is a class of neurological disorder ordisease, and where the neurological disease is characterized by agradual and progressive loss of neural tissue, and/or alteredneurological function, typically reduced neurological function as aresult of a gradual and progressive loss of neural tissue. Examples ofneurodegenerative diseases include for example, but are not limited to,Alzheimer's disease (AD), Parkinson's disease (PD), Huntington'sDisease, Amyotrophic Lateral Sclerosis (ALS, also termed Lou Gehrig'sdisease) and Multiple Sclerosis (MS), polyglutamine expansion disorders(e.g., HD, dentatorubropallidoluysian atrophy, Kennedy's disease (alsoreferred to as spinobulbar muscular atrophy), spinocerebellar ataxia(e.g., type 1, type 2, type 3 (also referred to as Machado-Josephdisease), type 6, type 7, and type 17)), other trinucleotide repeatexpansion disorders (e.g., fragile X syndrome, fragile XE mentalretardation, Friedreich's ataxia, myotonic dystrophy, spinocerebellarataxia type 8, and spinocerebellar ataxia type 12), Alexander disease,Alper's disease, ataxia telangiectasia, Batten disease (also referred toas Spielmeyer-Vogt-Sjogren-Batten disease), Canavan disease, Cockaynesyndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, ischemiastroke, Krabbe disease, Lewy body dementia, multiple system atrophy,Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis,Refsum's disease, Sandhoff disease, Schilder's disease, spinal cordinjury, spinal muscular atrophy (SMA), SteeleRichardson-Olszewskidisease, Tabes dorsalis, and the like. In some embodiments, theneurodegenerative disease is Alzheimer's disease.

The expression or activity of CD33 can be reduced by inhibition of theexpression of CD33 protein or by inhibiting activity of CD33. Theexpression of CD33 can be inhibited by “gene silencing” methods commonlyknown by persons of ordinary skill in the art. The reduction in activitycan be due to affecting one or more characteristics of CD33 includingdecreasing its catalytic activity or by inhibition a co-factor of CD33or by binding to CD33 with a degree of avidity that is such that theoutcome is that beta amyloid uptake is increased in cells expressing theCD33, such microglial cells. In particular, inhibition of CD33 can bedetermined using an assay for measuring beta amyloid uptake in cellsexpressing CD33, for example, but not limited to, by using the CD33uptake assay as disclosed herein. In some embodiments, activity of CD33is sialic acid binding. Thus, the methods disclosed herein decrease,inhibit, or reduce binding of sialic acid to CD33.

The term “agent” refers to any entity which is normally not present ornot present at the levels being administered in the cell. Agent can beselected from a group comprising: chemicals; small molecules; nucleicacid sequences; nucleic acid analogues; proteins; peptides; aptamers;antibodies; or fragments thereof. A nucleic acid sequence can be RNA orDNA, and can be single or double stranded, and can be selected from agroup comprising; nucleic acid encoding a protein of interest,oligonucleotides, nucleic acid analogues, for example peptide-nucleicacid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA)etc. Such nucleic acid sequences include, for example, but are notlimited to, nucleic acid sequence encoding proteins, for example thatact as transcriptional repressors, antisense molecules, ribozymes, smallinhibitory nucleic acid sequences, for example but are not limited toRNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc.A protein and/or peptide or fragment thereof can be any protein ofinterest, for example, but are not limited to: mutated proteins;therapeutic proteins and truncated proteins, wherein the protein isnormally absent or expressed at lower levels in the cell. Proteins canalso be selected from a group comprising; mutated proteins, geneticallyengineered proteins, peptides, synthetic peptides, recombinant proteins,chimeric proteins, antibodies, midibodies, minibodies, triabodies,humanized proteins, humanized antibodies, chimeric antibodies, modifiedproteins and fragments thereof. Alternatively, the agent can beintracellular within the cell as a result of introduction of a nucleicacid sequence into the cell and its transcription resulting in theproduction of the nucleic acid and/or protein inhibitor of CD33 withinthe cell. In some embodiments, the agent is any chemical, entity ormoiety, including without limitation synthetic and naturally-occurringnon-proteinaceous entities. In certain embodiments the agent is a smallmolecule having a chemical moiety. For example, chemical moietiesincluded unsubstituted or substituted alkyl, aromatic, or heterocyclylmoieties including macrolides, leptomycins and related natural productsor analogues thereof. Agents can be known to have a desired activityand/or property, or can be selected from a library of diverse compounds.

In some embodiments, the agent can cross the blood-brain barrier or canbe formulated to cross the blood-brain barrier.

In some embodiments, the agent that inhibits the expression or activityof CD33 is a nucleic acid, also referred to as a nucleic acid agentherein. In the context of this disclosure, the term “nucleic acid”refers to a polymer or oligomer of nucleotide or nucleoside monomersconsisting of naturally occurring bases, sugars and intersugar linkages.The term “nucleic acid” or “oligonucleotide” or “polynucleotide” areused interchangeably herein and can mean at least two nucleotidescovalently linked together. As will be appreciated by those in the art,the depiction of a single strand also defines the sequence of thecomplementary strand. Thus, a nucleic acid also encompasses thecomplementary strand of a depicted single strand. As will also beappreciated by those in the art, many variants of a nucleic acid can beused for the same purpose as a given nucleic acid. Thus, a nucleic acidalso encompasses substantially identical nucleic acids and complementsthereof.

Without limitation, the nucleic acid agent can be single-stranded ordouble-stranded. A single-stranded nucleic acid agent can havedouble-stranded regions and a double-stranded nucleic acid agent canhave single-stranded regions. The nucleic acid can be of any desiredlength. In particular embodiments, nucleic acid can range from about 10to 100 nucleotides in length. In various related embodiments, nucleicacid agents, single-stranded, double-stranded, and triple-stranded, canrange in length from about 10 to about 50 nucleotides, from about 20 toabout 50 nucleotides, from about 15 to about 30 nucleotides, from about20 to about 30 nucleotides in length. In some embodiments, nucleic acidagent is from about 9 to about 39 nucleotides in length. In some otherembodiments, nucleic acid agent is at least 30 nucleotides in length.

The nucleic acid agent can comprise any nucleic acid or oligonucleotidemodification described herein and below. In certain instances, it can bedesirable to modify one or both strands of a double-stranded nucleicacid agent. In some cases, the two strands will include differentmodifications. In other instances, multiple different modifications canbe included on each of the strands. The various modifications on a givenstrand can differ from each other, and can also differ from the variousmodifications on other strands. For example, one strand can have amodification, e.g., a modification described herein, and a differentstrand can have a different modification, e.g., a different modificationdescribed herein. In other cases, one strand can have two or moredifferent modifications, and the another strand can include amodification that differs from the at least two modifications on thefirst strand.

Single-stranded and double-stranded nucleic acid agents that areeffective in inducing RNA interference are referred to as siRNA, RNAiagent, iRNA agent, or RNAi inhibitor herein. As used herein, the term“iRNA agent” refers to a nucleic acid agent which can mediate thetargeted cleavage of an RNA transcript via an RNA-induced silencingcomplex (RISC) pathway.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a CD33 gene, including messenger (mRNA) that is a product of RNAprocessing of a primary transcription product. The target portion of thesequence will be at least long enough to serve as a substrate foriRNA-directed cleavage or binding of antisense RNA/oligonucleotide at ornear that portion. For example, the target sequence will generally befrom 9-36 nucleotides in length, e.g., 15-30 nucleotides in length,including all sub-ranges therebetween. As non-limiting examples, thetarget sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides,15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides,18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides,19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides,20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.

While a target sequence is generally 15-30 nucleotides in length, thereis wide variation in the suitability of particular sequences in thisrange for directing cleavage of any given target RNA. Various softwarepackages and the guidelines set out herein provide guidance for theidentification of optimal target sequences for any given gene target,but an empirical approach can also be taken in which a “window” or“mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that canserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression.Further, the target sequence can start at any desired nucleotideposition of the given target RNA.

CD33 mRNA sequences are known in the art. For example, human CD33 mRNAsequence can be accessed from NCBI Refseq collection by NCBI ReferenceSequences: NM_(—)001082618.1 (SEQ ID NO: 3); NM_(—)001772.3 (SEQ ID NO:4); NM_(—)001177608.1 (SEQ ID NO: 5); XM_(—)005259434.1 (SEQ ID NO: 6);and XM_(—)005259433.1 (SEQ ID NO: 7). Without limitations, the targetsequence can be a portion of any one of the above noted human CD33 mRNAsequences. In some embodiments, the nucleic acid agent comprises anucleotide substantially complementary to 15-30 nucleotides, 15-26nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides,15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides,18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides,19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides,20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or21-22 nucleotides contiguous nucleotides of one of the above-noted humanCD33 mRNA sequences.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as can beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

Complementary sequences within a double-stranded nucleic acid includebase-pairing of the oligonucleotide or polynucleotide comprising a firstnucleotide sequence to an oligonucleotide or polynucleotide comprising asecond nucleotide sequence over the entire length of one or bothnucleotide sequences. Such sequences can be referred to as “fullycomplementary” with respect to each other herein. However, where a firstsequence is referred to as “substantially complementary” with respect toa second sequence herein, the two sequences can be fully complementary,or they can form one or more, but generally not more than 5, 4, 3 or 2mismatched base pairs upon hybridization for a duplex up to 30 basepairs (bp), while retaining the ability to hybridize under theconditions most relevant to their ultimate application, e.g., inhibitionof gene expression via a RISC pathway. However, where twooligonucleotides are designed to form, upon hybridization, one or moresingle stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, can yet be referred to as“fully complementary” for the purposes described herein.

A percent complementarity indicates the percentage of contiguousresidues in a nucleic acid molecule that can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” or 100% complementarity meansthat all the contiguous residues of a nucleic acid sequence willhydrogen bond with the same number of contiguous residues in a secondnucleic acid sequence. Less than perfect complementarity refers to thesituation in which some, but not all, nucleoside units of two strandscan hydrogen bond with each other. “Substantial complementarity” refersto polynucleotide strands exhibiting 90% or greater complementarity,excluding regions of the polynucleotide strands, such as overhangs, thatare selected so as to be noncomplementary. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.The non-target sequences typically differ by at least 5 nucleotides.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” an mRNA refers to a polynucleotide that issubstantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding CD33). For example, a polynucleotide iscomplementary to at least a part of a CD33 mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding CD33.

The term “double-stranded RNA” or “dsRNA,” as used herein refers to aniRNA agent that includes an RNA molecule or complex of molecules havinga hybridized duplex region that comprises having “sense” and “antisense”orientations with respect to a target RNA. The duplex region can be ofany length that permits specific degradation of a desired target RNAthrough a RISC pathway, but will typically range from 9 to 36 base pairs(bp) in length, e.g., 15-30 bp in length. Considering a duplex between 9and 36 bp the duplex can be any length in this range, for example, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range therein between,including, but not limited to 15-30 bp, 15-26 bp, 15-23 bp, 15-22 bp,15-21 bp, 15-20 bp, 15-19 bp, 15-18 bp, 15-17 bp, 18-30 bp, 18-26 bp,18-23 bp, 18-22 bp, 18-21 bp, 18-20 bp, 19-30 bp, 19-26 bp, 19-23 bp,19-22 bp, 19-21 bp, 19-20 bp, 20-30 bp, 20-26 bp, 20-25 bp, 20-24 bp,20-23 bp, 20-22 bp, 20-21 bp, 21-30 bp, 21-26 bp, 21-25 bp, 21-24 bp,21-23 bp, or 21-22 bp. dsRNAs generated in the cell by processing withDicer and similar enzymes are generally in the range of 19-22 bp inlength. One strand of the duplex region of a dsDNA comprises a sequencethat is substantially complementary to a region of a target RNA. The twostrands forming the duplex structure can be from a single RNA moleculehaving at least one self-complementary region, or can be formed from twoor more separate RNA molecules. Where the duplex region is formed fromtwo strands of a single molecule, the molecule can have a duplex regionseparated by a single stranded chain of nucleotides (herein referred toas a “hairpin loop”) between the 3′-end of one strand and the 5′-end ofthe respective other strand forming the duplex structure. The hairpinloop can comprise at least one unpaired nucleotide; in some embodimentsthe hairpin loop can comprise at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 20,at least 23 or more unpaired nucleotides. Where the two substantiallycomplementary strands of a dsRNA are comprised by separate RNAmolecules, those molecules need not, but can be covalently connected.Where the two strands are connected covalently by means other than ahairpin loop, the connecting structure is referred to as a “linker.” Theterm “siRNA” is also used herein to refer to a dsRNA as described above.

In one aspect, an RNA interference agent includes a single stranded RNAthat interacts with a target RNA sequence to direct the cleavage of thetarget RNA. Without wishing to be bound by theory, long double strandedRNA introduced into plants and invertebrate cells is broken down intosiRNA by a Type III endonuclease known as Dicer (Sharp et al., GenesDev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes thedsRNA into 19-23 base pair short interfering RNAs with characteristictwo base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). ThesiRNAs are then incorporated into an RNA-induced silencing complex(RISC) where one or more helicases unwind the siRNA duplex, enabling thecomplementary antisense strand to guide target recognition (Nykanen, etal., (2001) Cell 107:309). Upon binding to the appropriate target mRNA,one or more endonucleases within the RISC cleaves the target to inducesilencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in oneaspect the invention relates to a single stranded RNA that promotes theformation of a RISC complex to effect silencing of the target gene.

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′ end, 3′ end orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotideoverhang at the 3′ end and/or the 5′ end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/orthe 5′ end. In another embodiment, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to adsRNA mean that there are no unpaired nucleotides or nucleotide analogsat a given terminal end of a dsRNA, i.e., no nucleotide overhang. One orboth ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt,the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNAis a dsRNA that is blunt at both ends, i.e., no nucleotide overhang ateither end of the molecule. Most often such a molecule will bedouble-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence. As used herein, the term “region ofcomplementarity” refers to the region on the antisense strand that issubstantially complementary to a sequence, for example a targetsequence, as defined herein. Where the region of complementarity is notfully complementary to the target sequence, the mismatches can be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

The double stranded RNAs can also include double-strandedoligonucleotide wherein the two strands are linked together. The twostrands can be linked to each other at both ends, or at one end only. Bylinking at one end is meant that 5′-end of first strand is linked to the3′-end of the second strand or 3′-end of first strand is linked to5′-end of the second strand. When the two strands are linked to eachother at both ends, 5′-end of first strand is linked to 3′-end of secondstrand and 3′-end of first strand is linked to 5′-end of second strand.The two strands can be linked together by an oligonucleotide linkerincluding, but not limited to, (N)_(n); wherein N is independently amodified or unmodified nucleotide and n is 3-23. In some embodiments, nis 3-10. In some embodiments, the oligonucleotide linker is (dT)₄ or(U)₄.

Hairpin and dumbbell type RNAi agents will have a duplex region equal toor less than 200, 100, or 50 nucleotides in length. In some embodiments,ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21nucleotides pairs in length. In some embodiments, the hairpinoligonucleotides can mimic the natural precursors of microRNAs. Thehairpin RNAi agents can have a single strand overhang or terminalunpaired region, in some embodiments at the 3′, and in some embodimentson the antisense side of the hairpin. In some embodiments, the overhangsare 1-4, more generally 2-3 nucleotides in length. The two strandsmaking up the hairpin structure can be arranged in any orientation. Forexample, the 3′-end of the antisense strand can be linked to the 5′-endof the sense strand, or the 5′-end of the antisense strand can be linkedto the 3′-end of the sense strand. The hairpin oligonucleotides are alsoreferred to as “shRNA” herein.

In some embodiments, the agent that inhibits the expression or activityof CD33 is an antisense oligonucleotide. One of skill in the art is wellaware that single-stranded oligonucleotides can hybridize to acomplementary target sequence and prevent access of the translationmachinery to the target RNA transcript, thereby preventing proteinsynthesis. The single-stranded oligonucleotide can also hybridize to acomplementary RNA and the RNA target can be subsequently cleaved by anenzyme such as RNase H and thus preventing translation of target RNA.Alternatively, or in addition to, the single-stranded oligonucleotidecan modulate the expression of a target sequence via RISC mediatedcleavage of the target sequence, i.e., the single-strandedoligonucleotide acts as a single-stranded RNAi agent. A “single-strandedRNAi agent” as used herein, is an RNAi agent which is made up of asingle molecule. A single-stranded RNAi agent can include a duplexedregion, formed by intra-strand pairing, e.g., it can be, or include, ahairpin or pan-handle structure.

In some embodiments, the agent that inhibits the expression or activityof CD33 is a microRNA. MicroRNAs (miRNAs or mirs) are a highly conservedclass of small RNA molecules that are transcribed from DNA in thegenomes of plants and animals, but are not translated into protein.Pre-microRNAs are processed into miRNAs. Processed microRNAs are singlestranded ˜17-25 nucleotide (nt) RNA molecules that become incorporatedinto the RNA-induced silencing complex (RISC) and have been identifiedas key regulators of development, cell proliferation, apoptosis anddifferentiation. They are believed to play a role in regulation of geneexpression by binding to the 3′-untranslated region of specific mRNAs.RISC mediates down-regulation of gene expression through translationalinhibition, transcript cleavage, or both. RISC is also implicated intranscriptional silencing in the nucleus of a wide range of eukaryotes.

The number of miRNA sequences identified to date is large and growing,illustrative examples of which can be found, for example, in: “miRBase:microRNA sequences, targets and gene nomenclature” Griffiths-Jones S,Grocock R J, van Dongen S, Bateman A, Enright A J. NAR, 2006, 34,Database Issue, D140-D144; “The microRNA Registry” Griffiths-Jones S.NAR, 2004, 32, Database Issue, D109-D111; and also on the worldwide webat http://microrna.dot.sanger.dot.ac.dot.uk/sequences/.

MiRNA mimics represent oligonucleotides that can be used to imitate thegene modulating activity of one or more miRNAs. Thus, the term “microRNAmimic” refers to synthetic non-coding RNAs (i.e. the miRNA is notobtained by purification from a source of the endogenous miRNA) that arecapable of entering the RNAi pathway and regulating gene expression.miRNA mimics can be designed as mature molecules (e.g. single stranded)or mimic precursors (e.g., pri- or pre-miRNAs). In one design, miRNAmimics are double stranded molecules (e.g., with a duplex region ofbetween about 16 and about 31 nucleotides in length) and contain one ormore sequences that have identity with the mature strand of a givenmiRNA. Double-stranded miRNA mimics have designs similar to as describedabove for double-stranded oligonucleotides.

In some embodiments, the agent that inhibits the expression or activityof CD33 is a ribozyme. Ribozymes are oligonucleotides having specificcatalytic domains that possess endonuclease activity (Kim and Cech, ProcNatl Acad Sci USA. 1987 December; 84(24):8788-92; Forster and Symons,Cell. 1987 Apr. 24; 49(2):211-20). At least six basic varieties ofnaturally-occurring enzymatic RNAs are known presently. In general,enzymatic nucleic acids act by first binding to a target RNA. Suchbinding occurs through the target binding portion of an enzymaticnucleic acid which is held in close proximity to an enzymatic portion ofthe molecule that acts to cleave the target RNA. Thus, the enzymaticnucleic acid first recognizes and then binds a target RNA throughcomplementary base-pairing, and once bound to the correct site, actsenzymatically to cut the target RNA. Strategic cleavage of such a targetRNA will destroy its ability to direct synthesis of an encoded protein.After an enzymatic nucleic acid has bound and cleaved its RNA target, itis released from that RNA to search for another target and canrepeatedly bind and cleave new targets.

Methods of producing a ribozyme targeted to any target sequence areknown in the art. Ribozymes can be designed as described in Int. Pat.Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595,each specifically incorporated herein by reference, and synthesized tobe tested in vitro and in vivo, as described therein.

Because transcription factors recognize their relatively short bindingsequences, even in the absence of surrounding genomic DNA, shortoligonucleotides bearing the consensus binding sequence of a specifictranscription factor can be used as tools for manipulating geneexpression in living cells. This strategy involves the intracellulardelivery of such “decoy oligonucleotides”, which are then recognized andbound by the target factor. Occupation of the transcription factor'sDNA-binding site by the decoy renders the transcription factor incapableof subsequently binding to the promoter regions of target genes. Decoyscan be used as therapeutic agents, either to inhibit the expression ofgenes that are activated by a transcription factor, or to up-regulategenes that are suppressed by the binding of a transcription factor.Examples of the utilization of decoy oligonucleotides can be found inMann et al., J. Clin. Invest., 2000, 106: 1071-1075, which is expresslyincorporated by reference herein, in its entirety.

The terms “antimir” “microRNA inhibitor” or “miR inhibitor” aresynonymous and refer to oligonucleotides that interfere with theactivity of specific miRNAs Inhibitors can adopt a variety ofconfigurations including single stranded, double stranded (RNA/RNA orRNA/DNA duplexes), and hairpin designs, in general, microRNA inhibitorscomprise one or more sequences or portions of sequences that arecomplementary or partially complementary with the mature strand (orstrands) of the miRNA to be targeted, in addition, the miRNA inhibitorcan also comprise additional sequences located 5′ and 3′ to the sequencethat is the reverse complement of the mature miRNA. The additionalsequences can be the reverse complements of the sequences that areadjacent to the mature miRNA in the pri-miRNA from which the maturemiRNA is derived, or the additional sequences can be arbitrary sequences(having a mixture of A, G, C, U, or dT). In some embodiments, one orboth of the additional sequences are arbitrary sequences capable offorming hairpins. Thus, in some embodiments, the sequence that is thereverse complement of the miRNA is flanked on the 5′ side and on the 3′side by hairpin structures. MicroRNA inhibitors, when double stranded,can include mismatches between nucleotides on opposite strands.

MicroRNA inhibitors, including hairpin miRNA inhibitors, are describedin detail in Vermeulen et al., “Double-Stranded Regions Are EssentialDesign Components Of Potent Inhibitors of RISC Function,” RNA 13:723-730 (2007) and in WO2007/095387 and WO 2008/036825 each of which isincorporated herein by reference in its entirety. A person of ordinaryskill in the art can select a sequence from the database for a desiredmiRNA and design an inhibitor useful for the methods disclosed herein.

Unmodified oligonucleotides can be less than optimal in someapplications, e.g., unmodified oligonucleotides can be prone todegradation by e.g., cellular nucleases. However, chemical modificationsto one or more of the subunits of oligonucleotide can confer improvedproperties, e.g., can render oligonucleotides more stable to nucleases.Typical oligonucleotide modifications can include one or more of: (i)alteration, e.g., replacement, of one or both of the non-linkingphosphate oxygens and/or of one or more of the linking phosphate oxygensin the phosphodiester intersugar linkage; (ii) alteration, e.g.,replacement, of a constituent of the ribose sugar, e.g., of themodification or replacement of the 2′ hydroxyl on the ribose sugar;(iii) wholesale replacement of the phosphate moiety; (iv) modificationor replacement of a naturally occurring base with a non-natural base;(v) replacement or modification of the ribose-phosphate backbone, e.g.with peptide nucleic acid (PNA); (vi) modification of the 3′ end or 5′end of the oligonucleotide; and (vii) modification of the sugar, e.g.,six membered rings.

The terms replacement, modification, alteration, and the like, as usedin this context, do not imply any process limitation, e.g., modificationdoes not mean that one must start with a reference or naturallyoccurring ribonucleic acid and modify it to produce a modifiedribonucleic acid bur rather modified simply indicates a difference froma naturally occurring molecule. As described below, modifications, e.g.,those described herein, can be provided as asymmetrical modifications.

The phosphate group in the intersugar linkage can be modified byreplacing one of the oxygens with a different substituent. One result ofthis modification to RNA phosphate intersugar linkages can be increasedresistance of the oligonucleotide to nucleolytic breakdown. Examples ofmodified phosphate groups include phosphorothioate, phosphoroselenates,borano phosphates, borano phosphate esters, hydrogen phosphonates,phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. Insome embodiments, one of the non-bridging phosphate oxygen atoms in theintersugar linkage can be replaced by a sulfur atom, e.g.phosphorothioates and phosphorodithioates. Phosphorodithioates have bothnon-bridging oxygens replaced by sulfur. The phosphorus center in thephosphorodithioates is achiral which precludes the formation ofoligonucleotides diastereomers.

The phosphate linker can also be modified by replacement of bridgingoxygen, (i.e. oxygen that links the phosphate to the nucleoside), withnitrogen (bridged phosphoroamidates), sulfur (bridgedphosphorothioates), and carbon (bridged methylenephosphonates). Thereplacement can occur at the either one of the linking oxygens or atboth linking oxygens. When the bridging oxygen is the 3′-oxygen of anucleoside, replacement with carbon is preferred. When the bridgingoxygen is the 5′-oxygen of a nucleoside, replacement with nitrogen ispreferred.

Modified phosphate linkages where at least one of the oxygen linked tothe phosphate has been replaced or the phosphate group has been replacedby a non-phosphorous group, are also referred to as “non-phosphodiesterintersugar linkage” or “non-phosphodiester linker.”

The phosphate group can be replaced by non-phosphorus containingconnectors, e.g. dephospho linkers. Dephospho linkers are also referredto as non-phosphodiester linkers herein. Examples of moieties which canreplace the phosphate group include, but are not limited to, amides,hydroxylamino, siloxanes, carboxamide, formacetal, methylenemethylimino(MMI). Further examples of dephospho linkers are described, for examplein, Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi andP. D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65),content of which is herein incorporated by reference.

The canonical 3′-5′ intersugar linkage can be replaced with a 5′-5′,3′-3′, 3′-2′, 2′-5′, 2′-3′ or 2′-2′ intersugar linkage.

An oligonucleotide can include modification of all or some of the sugargroups of the nucleic acid. For example, the 2′-position of the ribosesugar can be modified, e.g., the 2′-hydroxyl group (OH) can be modifiedor replaced with a number of different substituents. While not beingbound by theory, enhanced stability is expected since the hydroxyl canno longer be deprotonated to form a 2′-alkoxide ion. The 2′-alkoxide cancatalyze degradation by intramolecular nucleophilic attack on the linkerphosphorus atom. Again, while not wishing to be bound by theory, it canbe desirable to some embodiments to introduce alterations in whichalkoxide formation at the 2′-position is not possible. Exemplarymodifications at the 2′-position include, but are not limited to,2′-O-Me (2′-O-methyl), 2′-O-MOE (2′-O-methoxyethyl), 2′-halo (e.g.,2′-F), 2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), 2′-S-methyl,2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), 2′-CN, 2′-NH₂, 2′-SH, and “locked” nucleic acids (LNA),in which the oxygen at the 2′ position is connected by (CH₂)_(n),wherein n=1-4, to the 4′ carbon of the same ribose sugar, preferably nis 1 (LNA) or 2 (ENA).

Oligonucleotides can also include abasic sugars, which lack a nucleobaseat C-1′ or has other chemical groups in place of a nucleobase at C1′.See for example U.S. Pat. No. 5,998,203, content of which is hereinincorporated in its entirety by reference. Oligonucleotides can alsocontain one or more sugars that are the L isomer, e.g. L-nucleosides.Modification to the sugar group can also include replacement of the 4′-Owith a sulfur, optionally substituted nitrogen or CH₂ group.

Oligonucleotide modifications can also include acyclic nucleotides,wherein a C—C bonds between ribose carbons (e.g., C1′-C2′, C2′-C3′,C3′-C4′, C4′-O4′, C1′-O4′) is absent and/or at least one of ribosecarbons or oxygen (e.g., C1′, C2′, C3′, C4′ or O4′) are independently orin combination absent from the nucleotide.

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.For example, the 3′ and/or 5′ ends of an oligonucleotide can beconjugated to other functional molecular entities such as labeling m

An oligonucleotide can also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. When a naturalbase is replaced by a non-natural and/or universal base, the nucleotideis said to comprise a modified nucleobase and/or a nucleobasemodification herein. As used herein, “unmodified” or “natural”nucleobases include the purine bases adenine (A) and guanine (G), andthe pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modifiednucleobases include, but are not limited to, those described in U.S.Pat. No. 3,687,808; those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990; those disclosed by English et al., AngewandteChemie, International Edition, 1991, 30, 613; those disclosed inModified Nucleosides in Biochemistry, Biotechnology and Medicine,Herdewijin, P. Ed. Wiley-VCH, 2008; and those disclosed by Sanghvi, Y.S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Eds., CRC Press, 1993. Contents of all of theabove are herein incorporated by reference. As used herein, a universalnucleobase is any modified or nucleobase that can base pair with all ofthe four naturally occurring nucleobases without substantially affectingthe melting behavior, recognition by intracellular enzymes or activityof the oligonucleotide duplex. Some exemplary universal nucleobasesinclude, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl,nitroindolyl, 8-aza-7-deazaadenine, and structural derivatives thereof.See for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447,content of which is herein incorporated by reference.

In vivo applications of oligonucleotides is limited due to presence ofnucleases in the serum and/or blood. Thus in certain instances it ispreferable to modify the 3′, 5′ or both ends of an oligonucleotide tomake the oligonucleotide resistant against exonucleases. In someembodiments, the oligonucleotide comprises a cap structure at 3′(3′-cap), 5′ (5′-cap) or both ends. In some embodiments, oligonucleotidecomprises a 3′-cap. In another embodiment, oligonucleotide comprises a5′-cap. In yet another embodiment, oligonucleotide comprises both a 3′cap and a 5′ cap. It is to be understood that when an oligonucleotidecomprises both a 3′ cap and a 5′ cap, such caps can be same or they canbe different. As used herein, “cap structure” refers to chemicalmodifications, which have been incorporated at either terminus ofoligonucleotide. See for example U.S. Pat. No. 5,998,203 andInternational Patent Publication WO03/70918, contents of which areherein incorporated in their entireties.

Exemplary 5′-caps include, but are not limited to, 5′-5′-invertednucleotide, 5′-5′-inverted abasic nucleotide residue, and 2′-5′ linkage.Exemplary 3′-caps include, but are not limited to, ligands,3′-3′-inverted nucleotide, 3′-3′-inverted abasic nucleotide residue,3′-2′-inverted nucleotide moiety, 3′-2′-inverted abasic moiety, and2′-5′-linkage. For more details see Beaucage and Iyer, 1993, Tetrahedron49, 1925, incorporated by reference herein.

The oligonucleotides used in accordance with this invention can besynthesized with solid phase synthesis, see for example “Oligonucleotidesynthesis, a practical approach”, Ed. M. J. Gait, IRL Press, 1984;“Oligonucleotides and Analogues, A Practical Approach”, Ed. F. Eckstein,IRL Press, 1991 (especially Chapter 1, Modern machine-aided methods ofoligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotidesynthesis, Chapter 3, 2′-0-Methyloligoribonucleotides: synthesis andapplications, Chapter 4, Phosphorothioate oligonucleotides, Chapter 5,Synthesis of oligonucleotide phosphorodithioates, Chapter 6, Synthesisof oligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein. Modification described inWO 00/44895, WO01/75164, or WO02/44321 can be used herein. Thedisclosure of all publications, patents, and published patentapplications listed herein are hereby incorporated by reference.

The preparation of phosphinate oligonucleotides is described in U.S.Pat. No. 5,508,270. The preparation of alkyl phosphonateoligonucleotides is described in U.S. Pat. No. 4,469,863. Thepreparation of phosphoramidite oligonucleotides is described in U.S.Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation ofphosphotriester oligonucleotides is described in U.S. Pat. No.5,023,243. The preparation of boranophosphate oligonucleotide isdescribed in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of3′-Deoxy-3′-amino phosphoramidate oligonucleotides is described in U.S.Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonate oligonucleotides isdescribed in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801.Preparation of sulfur bridged nucleotides is described in Sproat et al.Nucleosides Nucleotides 1988, 7,651 and Crosstick et al. TetrahedronLett. 1989, 30, 4693.

Modifications to the 2′ modifications can be found in Verma, S. et al.Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein.Specific modifications to the ribose can be found in the followingreferences: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36,831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Methylenemethylimino linked oligonucleosides, also identified herein asMMI linked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified herein as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identifiedherein as amide-3 linked oligonucleosides, and methyleneaminocarbonyllinked oligonucleosides, also identified herein as amide-4 linkedoligonucleosides as well as mixed intersugar linkage compounds having,as for instance, alternating MMI and PO or PS linkages can be preparedas is described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and inInternational Application Nos. PCT/US92/04294 and PCT/US92/04305.Formacetal and thioformacetal linked oligonucleosides can be prepared asis described in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxidelinked oligonucleosides can be prepared as is described in U.S. Pat. No.5,223,618. Siloxane replacements are described in Cormier, J. F. et al.Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements are describedin Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethylreplacements are described in Edge, M. D. et al. J. Chem. Soc. PerkinTrans. 1 1972, 1991. Carbamate replacements are described in Stirchak,E. P. Nucleic Acids Res. 1989, 17, 6129.

Cyclobutyl sugar surrogate compounds can be prepared as is described inU.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared asis described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates canbe prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033,and other related patent disclosures. Peptide Nucleic Acids (PNAs) areknown per se and can be prepared in accordance with any of the variousprocedures referred to in Peptide Nucleic Acids (PNA): Synthesis,Properties and Potential Applications, Bioorganic & Medicinal Chemistry,1996, 4, 5-23. They can also be prepared in accordance with U.S. Pat.No. 5,539,083.

Terminal modifications are described in Manoharan, M. et al. Antisenseand Nucleic Acid Drug Development 12, 103-128 (2002) and referencestherein, content of which is herein incorporated by reference in itsentirety.

Representative U.S. patents that teach the preparation of certainmodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,457,191;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886;6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640;6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, content ofeach of which is herein incorporated by reference in its entirety.

A wide variety of entities, e.g., ligands, can be coupled to theoligonucleotides described herein. Ligands can include naturallyoccurring molecules, or recombinant or synthetic molecules. Exemplaryligands include, but are not limited to, peptides, peptidomimetics,polylysine (PLL), polyethylene glycol (PEG), mPEG, cationic groups,spermine, spermidine, polyamine, thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, mucin, glycosylated polyaminoacids,transferrin, aptamer, immunoglobulins (e.g., antibodies), insulin,transferrin, albumin, sugar, lipophilic molecules (e.g, steroids, bileacids, cholesterol, cholic acid, and fatty acids), vitamin A, vitamin E,vitamin K, vitamin B, folic acid, B12, riboflavin, biotin, pyridoxal,vitamin cofactors, lipopolysaccharide, hormones and hormone receptors,lectins, carbohydrates, multivalent carbohydrates, radiolabeled markers,fluorescent dyes, and derivatives thereof. Representative U.S. patentsthat teach the preparation of oligonucleotide conjugates include, butare not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218, 105;5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578, 717, 5,580,731;5,580,731; 5,591,584; 5,109,124; 5,118, 802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578, 718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762, 779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904, 582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082, 830;5,112,963; 5,149,782; 5,214,136; 5,245,022; 5,254, 469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510, 475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574, 142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599, 923; 5,599,928; 5,672,662; 5,688,941; 5,714,166; 6,153, 737;6,172,208; 6,300,319; 6,335,434; 6,335,437; 6,395, 437; 6,444,806;6,486,308; 6,525,031; 6,528,631; 6,559, 279; contents which are hereinincorporated in their entireties by reference.

In general, any method of delivering a nucleic acid molecule can beadapted for use with the nucleic acid agents described herein (see e.g.,Akhtar S, and Julian R L., 1992, Trends Cell. Biol. 2 (5):139-144 andWO94/02595, which are incorporated herein by reference in theirentireties). However, there are three factors that are important toconsider in order to successfully delivering a nucleic acid agent invivo: (a) biological stability of the delivered molecule, (2) preventingnon-specific effects, and (3) accumulation of the delivered molecule inthe target tissue. The non-specific effects of a nucleic acid agent canbe minimized by local administration, for example by direct injection orimplantation into a tissue (as a non-limiting example, a tumor) ortopically administering the preparation. Local administration to atreatment site maximizes local concentration of the agent, limits theexposure of the agent to systemic tissues that may otherwise be harmedby the agent or that can degrade the agent, and permits a lower totaldose of the agent to be administered. Several studies have shownsuccessful knockdown of gene products when an iRNA agent is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering a nucleic acid agent systemically for the treatment of adisease, the nucleic acid agent can be modified or alternativelydelivered using a drug delivery system; both methods act to prevent therapid degradation of the nucleic acid agent by endo- and exo-nucleasesin vivo. In an alternative embodiment, the nucleic acid agent can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of anucleic acid molecule (negatively charged) and also enhance interactionsat the negatively charged cell membrane to permit efficient uptake ofthe nucleic acid agent by the cell. Cationic lipids, dendrimers, orpolymers can either be bound to a nucleic acid agent, or induced to forma vesicle or micelle (see e.g., Kim S H., et al (2008) Journal ofControlled Release 129 (2):107-116) that encases a nucleic acid agent.The formation of vesicles or micelles further prevents degradation ofthe nucleic acid agent when administered systemically. Methods formaking and administering cationic-nucleic acid complexes are well withinthe abilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol. 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of nucleic acids andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

In another aspect, a nucleic acid agent that inhibits the expression oractivity of CD33 can be expressed from transcription units inserted intoDNA or RNA vectors (see, e.g., Couture, A, et al., TIG., 1996, 12:5-10;Skillern, A., et al., International PCT Publication No. WO 00/22113,Conrad, PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No.6,054,299). Expression can be transient (on the order of hours to weeks)or sustained (weeks to months or longer), depending upon the specificconstruct used and the target tissue or cell type. These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be an integrating or non-integrating vector. Thetransgene can also be constructed to permit it to be inherited as anextrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA,1995, 92:1292).

The individual strand or strands of a dsRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

RNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell. Vectors are described below inmore detail.

In some embodiments, the agent that inhibits the expression or activityof CD33 is a small molecule. For example, irreversible or reversibleinhibitors of CD33 can be used in the methods disclosed herein. As usedherein, the term “small molecule” refers to natural or syntheticmolecules having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, than about 1,000 grams per mole, or lessthan about 500 grams per mole.

In some embodiments, the agent that inhibits the expression or activityof CD33 is a sialic acid analogue or a sialic acid derivative.

In some embodiments, the agent that inhibits the expression or activityof can be an antibody molecule or the epitope-binding moiety of anantibody molecule. For example the agent can be selected frommonoclonal, chimeric, humanized, and recombinant antibodies andantigen-binding fragments thereof. In some embodiments, neutralizingantibodies can be used as inhibitors of CD33. Antibodies are readilyraised in animals such as rabbits or mice by immunization with theantigen. Immunized mice are particularly useful for providing sources ofB cells for the manufacture of hybridomas, which in turn are cultured toproduce large quantities of monoclonal antibodies.

Antibodies provide high binding avidity and unique specificity to a widerange of target antigens and haptens. Monoclonal antibodies useful inthe practice of the methods disclosed herein include whole antibody andfragments thereof and are generated in accordance with conventionaltechniques, such as hybridoma synthesis, recombinant DNA techniques andprotein synthesis.

Useful monoclonal antibodies and fragments can be derived from anyspecies (including humans) or can be formed as chimeric proteins whichemploy sequences from more than one species. Human monoclonal antibodiesor “humanized” murine antibody are also used in accordance with thepresent invention. For example, murine monoclonal antibody can be“humanized” by genetically recombining the nucleotide sequence encodingthe murine Fv region (i.e., containing the antigen binding sites) or thecomplementarily determining regions thereof with the nucleotide sequenceencoding a human constant domain region and an Fc region. Humanizedtargeting moieties are recognized to decrease the immunoreactivity ofthe antibody or polypeptide in the host recipient, permitting anincrease in the half-life and a reduction the possibly of adverse immunereactions in a manner similar to that disclosed in European PatentApplication No. 0,411,893 A2. The murine monoclonal antibodies shouldpreferably be employed in humanized form. Antigen binding activity isdetermined by the sequences and conformation of the amino acids of thesix complementarily determining regions (CDRs) that are located (threeeach) on the light and heavy chains of the variable portion (Fv) of theantibody. The 25-kDa single-chain Fv (scFv) molecule, composed of avariable region (VL) of the light chain and a variable region (VH) ofthe heavy chain joined via a short peptide spacer sequence, is thesmallest antibody fragment developed to date. Techniques have beendeveloped to display scFv molecules on the surface of filamentous phagethat contain the gene for the scFv. scFv molecules with a broad range ofantigenic-specificities can be present in a single large pool ofscFv-phage library. Some examples of high affinity monoclonal antibodiesand chimeric derivatives thereof, useful in the methods of the presentinvention, are described in the European Patent Application EP 186,833;PCT Patent Application WO 92/16553; and U.S. Pat. No. 6,090,923.

Chimeric antibodies are immunoglobin molecules characterized by two ormore segments or portions derived from different animal species.Generally, the variable region of the chimeric antibody is derived froma non-human mammalian antibody, such as murine monoclonal antibody, andthe immunoglobin constant region is derived from a human immunoglobinmolecule. Preferably, both regions and the combination have lowimmunogenicity as routinely determined.

One limitation of scFv molecules is their monovalent interaction withtarget antigen. One of the easiest methods of improving the binding of ascFv to its target antigen is to increase its functional affinitythrough the creation of a multimer. Association of identical scFvmolecules to form diabodies, triabodies and tetrabodies can comprise anumber of identical Fv modules. These reagents are thereforemultivalent, but monospecific. The association of two different scFvmolecules, each comprising a VH and VL domain derived from differentparent Ig will form a fully functional bispecific diabody. A uniqueapplication of bispecific scFvs is to bind two sites simultaneously onthe same target molecule via two (adjacent) surface epitopes. Thesereagents gain a significant avidity advantage over a single scFv or Fabfragments. A number of multivalent scFv-based structures has beenengineered, including for example, miniantibodies, dimericminiantibodies, minibodies, (scFv)₂, diabodies and triabodies. Thesemolecules span a range of valence (two to four binding sites), size (50to 120 kDa), flexibility and ease of production. Single chain Fvantibody fragments (scFvs) are predominantly monomeric when the VH andVL domains are joined by, polypeptide linkers of at least 12 residues.The monomer scFv is thermodynamically stable with linkers of 12 and 25amino acids length under all conditions. The noncovalent diabody andtriabody molecules are easy to engineer and are produced by shorteningthe peptide linker that connects the variable heavy and variable lightchains of a single scFv molecule. The scFv dimers are joined byamphipathic helices that offer a high degree of flexibility and theminiantibody structure can be modified to create a dimeric bispecific(DiBi) miniantibody that contains two miniantibodies (four scFvmolecules) connected via a double helix. Gene-fused or disulfide bondedscFv dimers provide an intermediate degree of flexibility and aregenerated by straightforward cloning techniques adding a C-terminalGly4Cys sequence. scFv-CH3 minibodies are comprised of two scFvmolecules joined to an IgG CH3 domain either directly (LD minibody) orvia a very flexible hinge region (Flex minibody). With a molecularweight of approximately 80 kDa, these divalent constructs are capable ofsignificant binding to antigens. The Flex minibody exhibits impressivetumor localization in mice. Bi- and tri-specific multimers can be formedby association of different scFv molecules. Increase in functionalaffinity can be reached when Fab or single chain Fv antibody fragments(scFv) fragments are complexed into dimers, trimers or largeraggregates. The most important advantage of multivalent scFvs overmonovalent scFv and Fab fragments is the gain in functional bindingaffinity (avidity) to target antigens. High avidity requires that scFvmultimers are capable of binding simultaneously to separate targetantigens. The gain in functional affinity for scFv diabodies compared toscFv monomers is significant and is seen primarily in reduced off-rates,which result from multiple binding to two or more target antigens and torebinding when one Fv dissociates. When such scFv molecules associateinto multimers, they can be designed with either high avidity to asingle target antigen or with multiple specificities to different targetantigens. Multiple binding to antigens is dependent on correct alignmentand orientation in the Fv modules. For full avidity in multivalent scFvstarget, the antigen binding sites must point towards the same direction.If multiple binding is not sterically possible then apparent gains infunctional affinity are likely to be due the effect of increasedrebinding, which is dependent on diffusion rates and antigenconcentration. Antibodies conjugated with moieties that improve theirproperties are also contemplated for the instant invention. For example,antibody conjugates with PEG that increases their half-life in vivo canbe used for the present invention. Immune libraries are prepared bysubjecting the genes encoding variable antibody fragments from the Blymphocytes of naive or immunized animals or patients to PCRamplification. Combinations of oligonucleotides which are specific forimmunoglobulin genes or for the immunoglobulin gene families are used.Immunoglobulin germ line genes can be used to prepare semisyntheticantibody repertoires, with the complementarity-determining region of thevariable fragments being amplified by PCR using degenerate primers.These single-pot libraries have the advantage that antibody fragmentsagainst a large number of antigens can be isolated from one singlelibrary. The phage-display technique can be used to increase theaffinity of antibody fragments, with new libraries being prepared fromalready existing antibody fragments by random, codon-based orsite-directed mutagenesis, by shuffling the chains of individual domainswith those of fragments from naive repertoires or by using bacterialmutator strains.

Alternatively, a SCID-hu mouse, for example the model developed byGenpharm, can be used to produce antibodies, or fragments thereof. Inone embodiment, a new type of high avidity binding molecule, termedpeptabody, created by harnessing the effect of multivalent interactionis contemplated. A short peptide ligand was fused via a semirigid hingeregion with the coiled-coil assembly domain of the cartilage oligomericmatrix protein, resulting in a pentameric multivalent binding molecule.In preferred embodiment of this invention, ligands and/or chimericinhibitors can be targeted to tissue- or tumor-specific targets by usingbispecific antibodies, for example produced by chemical linkage of ananti-ligand antibody (Ab) and an Ab directed toward a specific target.To avoid the limitations of chemical conjugates, molecular conjugates ofantibodies can be used for production of recombinant bispecificsingle-chain Abs directing ligands and/or chimeric inhibitors at cellsurface molecules. Alternatively, two or more active agents and orinhibitors attached to targeting moieties can be administered, whereineach conjugate includes a targeting moiety, for example, a differentantibody. Each antibody is reactive with a different target site epitope(associated with the same or a different target site antigen). Thedifferent antibodies with the agents attached accumulate additively atthe desired target site. Antibody-based or non-antibody-based targetingmoieties can be employed to deliver a ligand or the inhibitor to atarget site. Preferably, a natural binding agent for an unregulated ordisease associated antigen is used for this purpose.

In some embodiments, the antibody comprises the amino acid sequence SEQID NO: 1.

In some embodiments, the methods disclosed herein comprise administeringa CD33 protein lacking the sialic acid binding domain (i.e., anon-sialic CD33 protein) to the subject. The Ig-V typically mediatessialic acid binding in CD33. Thus, the IG-V domain in the non-sialic CDpolypeptide is altered or modified such that the non-sialic CD33 proteindoes not bind sialic acid or the binding is decreased or reducedrelative to the full length CD33 protein. The Ig-V domain can becompletely absent in the non-sialic CD33 protein or comprises one ormore mutations and/or deletions such that its (Ig-V domain) ability tobind sialic acid is decreased, reduced or abolished.

Peptide modifications are well known in the art. Thus, a non-sialic CD33protein disclosed herein can comprise one or more peptide modificationsknown in the art. Exemplary peptide modifications for modifying thenon-sialic CD33 protein include, but are not limited to, D amino acids,α amino acids, β amino acids, non-amide or modified amide linkages,chemically modified amino acids, naturally occurring non-proteogenicamino acids, rare amino acids, chemically synthesized compounds thathave properties known in the art to be characteristic of an amino acid,and the like. In some embodiments the non-sialic CD33 protein comprisesat least one (e.g. two, three, four, five, six, seven, eight, nine, tenor more) peptide modifications.

In some embodiments, the non-sialic CD33 protein comprises at least one(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid selected fromthe group consisting of homocysteine, phosphoserine, phosphothreonine,phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid,octahydroindole-2-carboxylic acid, statine,1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine(3-mercapto-D-valine), ornithine, citruline, alpha-methyl-alanine,para-benzoylphenylalanine, para-amino phenylalanine,p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, andtert-butylglycine), diaminobutyric acid,7-hydroxy-tetrahydroisoquinoline carboxylic acid, naphthylalanine,biphenylalanine, cyclohexylalanine, amino-isobutyric acid, norvaline,norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid,pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine,dehydroleucine, 2,2-diethylglycine, 1-amino-1-cyclopentanecarboxylicacid, 1-amino-1-cyclohexanecarboxylic acid, amino-benzoic acid,amino-naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine,nipecotic acid, alpha-amino butyric acid, thienyl-alanine,t-butylglycine, and derivatives thereof.

In some embodiments, the non-sialic CD33 protein comprises at least one(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) D-amino acid. When morethan one D-amino acid is present in the non-sialic CD33 protein, theycan be positioned next to or not next to each other.

The non-sialic CD33 protein can also comprise one or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more) of beta-amino acids. When more than onebeta-amino acid is present in the non-sialic CD33 protein, they can bepositioned next to each other or next to another amino acid.

In some embodiments, the non-sialic CD33 protein comprises at least one(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) peptide bond replacementto make a more stable protein. Exemplary peptide bond replacementsinclude, but are not limited to, urea, thiourea, carbamate, sulfonylurea, trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid,para-(aminoalkyl)-phenylacetic acid, meta-(aminoalkyl)-phenylaceticacid, thioamide, tetrazole, boronic ester, and olefinic group. One ormore of the peptide bonds of in the non-sialic CD33 protein can bereplaced with a peptide bond replacement. The peptide bond can also bereplaced by a linker. When more than one peptide bond replacement ispresent in the non-sialic CD33 protein, they can be positioned next toeach other or next to unmodified peptide bond.

The choice of including a modification in the non-sialic CD33 proteindepends, in part, on the desired characteristics of the non-sialic CD33protein. For example, the incorporation of one or more (D)-amino acidscan confer increasing stability on the non-sialic CD33 protein in vitroor in vivo, thus affecting shelf-life, serum half-life orbioavailability. The incorporation of one or more (D)-amino acids alsocan increase or decrease the binding activity of the non-sialic CD33protein as determined, for example, using the binding assays by methodswell known in the art. In some cases it is desirable to design anon-sialic CD33 protein which retains activity for a longer period oftime, for example, when designing a peptide to administer to a subject.In these cases, the incorporation of one or more (D)-amino acids orreplacement of amide backbone linkages in the non-sialic CD33 proteincan stabilize the non-sialic CD33 protein against endogenous peptidasesin vivo, thereby prolonging the subject's exposure to the non-sialicCD33 protein.

The non-sialic CD33 proteins of the present invention can be synthesizedby using well known methods including recombinant methods and chemicalsynthesis. Recombinant methods of producing a peptide through theintroduction of a vector including nucleic acid encoding the peptideinto a suitable host cell is well known in the art, such as is describedin Sambrook et al., Molecular Cloning: A Laboratory Manual, 4^(th) Ed,Vols 1 to 3, Cold Spring Harbor, N.Y. (2012); M. W. Pennington and B. M.Dunn, Methods in Molecular Biology: Peptide Synthesis Protocols, Vol 35,Humana Press, Totawa, N.J. (1994), contents of both of which are hereinincorporated by reference. Peptides can also be chemically synthesizedusing methods well known in the art. See for example, Merrifield et al.,J. Am. Chem. Soc. 85:2149 (1964); Bodanszky, M., Principles of PeptideSynthesis, Springer-Verlag, New York, N.Y. (1984); Kimmerlin, T. andSeebach, D. J. Pept. Res. 65:229-260 (2005); Nilsson et al., Annu Rev.Biophys. Biomol. Struct. (2005) 34:91-118; W. C. Chan and P. D. White(Eds.) Fmoc Solid Phase Peptide Synthesis: A Practical Approach, OxfordUniversity Press, Cary, N.C. (2000); N. L. Benoiton, Chemistry ofPeptide Synthesis, CRC Press, Boca Raton, Fla. (2005); J. Jones, AminoAcid and Peptide Synthesis, 2^(nd) Ed, Oxford University Press, Cary,N.C. (2002); and P. Lloyd-Williams, F. Albericio, and E. Giralt,Chemical Approaches to the synthesis of peptides and proteins, CRCPress, Boca Raton, Fla. (1997), contents of all of which are hereinincorporated by reference. Peptide derivatives can also be prepared asdescribed in U.S. Pat. Nos. 4,612,302; 4,853,371; and 4,684,620, andU.S. Pat. App. Pub. No. 2009/0263843, contents of all which are hereinincorporated by reference.

Newly synthesized the non-sialic CD33 proteins can be purified, forexample, by high performance liquid chromatography (HPLC), and can becharacterized using, for example, mass spectrometry or amino acidsequence analysis.

In some embodiments, the non-sialic CD33 protein comprises the aminoacid sequence: mplllllpll wadlthrpki lipgtlepgh sknitcsysw aceqgtppifswlsaaptsl gprtthssvl iitprpqdhg tnitcqvkfa gagvtterti qlnvtyvpqnpttgifpgdg sgkqetragv vhgaiggagv tallalcicl iffivkthrr kaartavgrndthpttgsas pkhqkksklh gptetsscsg aaptvemdee lhyaslnfhg mnpskdtsteysevrtq (SEQ ID NO: 8).

In one embodiment, the non-sialic CD33 protein comprises an amino acidsequence SEQ ID NO: 2, wherein at least one amino acid is deleted,modified, or replaced by a different amino acid, such that binding ofsialic acid to the non-sialic CD33 protein is decreased, reduced orabolished.

In some embodiments, the non-sialic CD33 protein can be administered tothe subject by administering a nucleic acid encoding or expressing thenon-sialic CD33 protein to the subject. In one embodiment, the nucleicacid encoding the non-sialic CD33 protein comprises the nucleotidesequence:

(SEQ ID NO: 9) CAGACATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGACTTGACCCACAGGCCCAAAATCCTCATCCCTGGCACTCTAGAACCCGGCCACTCCAAAAACCTGACCTGCTCTGTGTCCTGGGCCTGTGAGCAGGGAACACCCCCGATCTTCTCCTGGTTGTCAGCTGCCCCCACCTCCCTGGGCCCCAGGACTACTCACTCCTCGGTGCTCATAATCACCCCACGGCCCCAGGACCACGGCACCAACCTGACCTGTCAGGTGAAGTTCGCTGGAGCTGGTGTGACTACGGAGAGAACCATCCAGCTCAACGTCACCTATGTTCCACAGAACCCAACAACTGGTATCTTTCCAGGAGATGGCTCAGGGAAACAAGAGACCAGAGCAGGAGTGGTTCATGGGGCCATTGGAGGAGCTGGTGTTACAGCCCTGCTCGCTCTTTGTCTCTGCCTCATCTTCTTCATAGTGAAGACCCACAGGAGGAAAGCAGCCAGGACAGCAGTGGGCAGGAATGACACCCACCCTACCACAGGGTCAGCCTCCCCGAAACACCAGAAGAAGTCCAAGTTACATGGCCCCACTGAAACCTCAAGCTGTTCAGGTGCCGCCCCTACTGTGGAGATGGATGAGGAGCTGCATTATGCTTCCCTCAACTTTCATGGGATGAATCCTTCCAAGGACACCTCCACCGAATACTCAGAGGTCAGGACCCAGTGAGGAACCCACAAGAGCATCAGGCTCAGCTAGAAGATCCACATCCTCTACAGGTCGGGGACCAAAGGCTGATTCTTGGAGATTTAACACCCCACAGGCAATGGGTTTATAGACATTATGTGAGTTTCCTGCTATATTAACATCATCTTAGACTTTGCAAGCAGAGAGTCGTGGAATCAAATCTGTGCTCTTTCATTTGCTAAGTGTATGATGTCACACAAGCTCCTTAACCTTCCATGTCTCCATTTTCTTCTCTGTGAAGTAGGTATAAGAAGTCCTATCTCATAGGGATGCTGTGAGCATTAAATAAAGGTACACATGGAAAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA.

In some embodiments, the nucleic acid encoding the non-sialic CD33protein is a modified RNA. It has been demonstrated that modified RNAcan be used for rapid and efficient protein expression endogenously. Inaddition, modified RNA is advantageous in that it can result in areduced innate immune response relative to a similar RNA without themodifications. Technologies for modified RNA are disclosed inWO2012138453 and US20120322865, the content of each of which isincorporated herein in its entirety.

In some embodiments, the nucleic acid encoding the non-sialic CD33protein is a vector that encodes and directs the expression of thenon-sialic CD33 protein.

As used herein, the term “vector” refers to a polynucleotide sequencesuitable for transferring transgenes into a host cell. The term “vector”includes plasmids, mini-chromosomes, phage, naked DNA and the like. See,for example, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828;5,888,783 and, 5,919,670, and, Sambrook et al, Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989). One type ofvector is a “plasmid,” which refers to a circular double stranded DNAloop into which additional DNA segments are ligated. Another type ofvector is a viral vector, wherein additional DNA segments are ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Moreover, certain vectors are capable of directingthe expression of genes to which they are operatively linked. Suchvectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” is used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

A cloning vector is one which is able to replicate autonomously orintegrated in the genome in a host cell, and which is furthercharacterized by one or more endonuclease restriction sites at which thevector may be cut in a determinable fashion and into which a desired DNAsequence can be ligated such that the new recombinant vector retains itsability to replicate in the host cell. In the case of plasmids,replication of the desired sequence can occur many times as the plasmidincreases in copy number within the host cell such as a host bacteriumor just a single time per host before the host reproduces by mitosis. Inthe case of phage, replication can occur actively during a lytic phaseor passively during a lysogenic phase.

An expression vector is one into which a desired DNA sequence can beinserted by restriction and ligation such that it is operably joined toregulatory sequences and can be expressed as an RNA transcript. Vectorscan further contain one or more marker sequences suitable for use in theidentification of cells which have or have not been transformed ortransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase, luciferase or alkaline phosphatase),and genes which visibly affect the phenotype of transformed ortransfected cells, hosts, colonies or plaques (e.g., green fluorescentprotein). In certain embodiments, the vectors used herein are capable ofautonomous replication and expression of the structural gene productspresent in the DNA segments to which they are operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably” joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript can be translated into thedesired protein or polypeptide.

The promoter can be a native promoter, i.e., the promoter of the gene inits endogenous context, which provides normal regulation of expressionof the gene. In some embodiments the promoter can be constitutive, i.e.,the promoter is unregulated allowing for continual transcription of itsassociated gene. A variety of conditional promoters also can be used,such as promoters controlled by the presence or absence of a molecule.

The precise nature of the regulatory sequences needed for geneexpression can vary between species or cell types, but in general caninclude, as necessary, 5′-non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. In particular, such 5′ non-transcribed regulatory sequenceswill include a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences can also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 4^(th)Ed, Vols 1 to 3, Cold Spring Harbor, N.Y. (2012).

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of CD33 protein in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of the non-sialic CD33 protein or thenucleic acid agent will include regulatory elements (promoter, enhancer,etc.) sufficient for expression of the CD33 protein or the nucleic acidagent in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the non-sialic CD33 protein or the nucleic acid can beprecisely regulated, for example, by using an inducible regulatorysequence that is sensitive to certain physiological regulators, e.g.,circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J.8:20-24). Such inducible expression systems, suitable for the control ofdsRNA expression in cells or in mammals include, for example, regulationby ecdysone, by estrogen, progesterone, tetracycline, chemical inducersof dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). Aperson skilled in the art would be able to choose the appropriateregulatory/promoter sequence based on the intended use of the iRNAtransgene.

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding the non-sialic CD33 protein or the nucleic acid agentcan be used. For example, a retroviral vector can be used. Theseretroviral vectors contain the components necessary for the correctpackaging of the viral genome and integration into the host cell DNA.More detail about retroviral vectors can be found, for example, inBoesen et al., Biotherapy 6:291-302 (1994), which describes the use of aretroviral vector to deliver the mdr1 gene to hematopoietic stem cellsin order to make the stem cells more resistant to chemotherapy. Otherreferences illustrating the use of retroviral vectors in gene therapyare: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al.,Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics andDevel. 3:110-114 (1993). Lentiviral vectors contemplated for useinclude, for example, the HIV based vectors described in U.S. Pat. Nos.6,143,520; 5,665,557; and 5,981,276, which are herein incorporated byreference.

Adenoviruses are also contemplated for use in delivery of non-sialicCD33 proteins or the nucleic acid agents. Adenoviruses are especiallyattractive vehicles, e.g., for delivering genes to respiratoryepithelia. Adenoviruses naturally infect respiratory epithelia wherethey cause a mild disease. Other targets for adenovirus-based deliverysystems are liver, the central nervous system, endothelial cells, andmuscle. Adenoviruses have the advantage of being capable of infectingnon-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics andDevelopment 3:499-503 (1993) present a review of adenovirus-based genetherapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated theuse of adenovirus vectors to transfer genes to the respiratory epitheliaof rhesus monkeys. Other instances of the use of adenoviruses in genetherapy can be found in Rosenfeld et al., Science 252:431-434 (1991);Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin.Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al.,Gene Therapy 2:775-783 (1995).

Use of Adeno-associated virus (AAV) vectors is also contemplated (Walshet al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No.5,436,146). In one embodiment, the CD33 protein can be expressed from arecombinant AAV vector having, for example, either the U6 or H1 RNApromoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectorsfor expressing the CD33 protein, methods for constructing therecombinant AV vector, and methods for delivering the vectors intotarget cells are described in Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski Ret al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S.Pat. No. 5,139,941; International Patent Application No. WO 94/13788;and International Patent Application No. WO 93/24641, the entiredisclosures of which are herein incorporated by reference.

Another preferred viral vector is a pox virus such as a vaccinia virus,for example an attenuated vaccinia such as Modified Virus Ankara (MVA)or NYVAC, an avipox such as fowl pox or canary pox.

The agents that inhibits the expression or activity of CD33 orexpression vectors can be provided in pharmaceutically acceptablecompositions. These pharmaceutically acceptable compositions comprise atherapeutically-effective amount of the agent formulated together withone or more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail below, the pharmaceutical compositionsof can be pecially formulated for administration in solid or liquidform, including those adapted for the following: (1) oraladministration, for example, drenches (aqueous or non-aqueous solutionsor suspensions), lozenges, dragees, capsules, pills, tablets (e.g.,those targeted for buccal, sublingual, and systemic absorption),boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; (8) transmucosally; or (9) nasally. Additionally,agents can be implanted into a patient or injected using a drug deliverysystem. See, for example, Urquhart, et al., Ann. Rev. Pharmacol.Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Release ofPesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S.Pat. No. 3,773,919; and U.S. Pat. No. 35 3,270,960.

As used here, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used here, the term “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment. Forexample, an amount of a compound administered to a subject that issufficient to produce a statistically significant, measurable change inat least one symptom of cancer or metastasis.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other pharmaceutically active agents.

Toxicity and therapeutic efficacy can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compositions that exhibit large therapeutic indices are preferred. Asused herein, the term ED denotes effective dose and is used inconnection with animal models. The term EC denotes effectiveconcentration and is used in connection with in vitro models.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized.

The therapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range that includes the 1050 (i.e., theconcentration of the therapeutic which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Levels in plasmacan be measured, for example, by high performance liquid chromatography.The effects of any particular dosage can be monitored by a suitablebioassay.

The dosage can be determined by a physician and adjusted, as necessary,to suit observed effects of the treatment. Generally, the compositionsare administered so that the agent is given at a dose from 1 μg/kg to150 mg/kg, 1 μg/kg to 100 mg/kg, 1 μg/kg to 50 mg/kg, 1 μg/kg to 20mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to 1 mg/kg, 100 μg/kg to 100 mg/kg,100 μg/kg to 50 mg/kg, 100 μg/kg to 20 mg/kg, 100 μg/kg to 10 mg/kg, 100μg/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50mg/kg, or 10 mg/kg to 20 mg/kg. It is to be understood that ranges givenhere include all intermediate ranges, for example, the range 1 tmg/kg to10 mg/kg includes 1 mg/kg to 2 mg/kg, 1 mg/kg to 3 mg/kg, 1 mg/kg to 4mg/kg, 1 mg/kg to 5 mg/kg, 1 mg/kg to 6 mg/kg, 1 mg/kg to 7 mg/kg, 1mg/kg to 8 mg/kg, 1 mg/kg to 9 mg/kg, 2 mg/kg to 10 mg/kg, 3 mg/kg to 10mg/kg, 4 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 6 mg/kg to 10 mg/kg, 7mg/kg to 10 mg/kg, 8 mg/kg to 10 mg/kg, 9 mg/kg to 10 mg/kg, and thelike. It is to be further understood that the ranges intermediate to thegiven above are also within the scope of this invention, for example, inthe range 1 mg/kg to 10 mg/kg, dose ranges such as 2 mg/kg to 8 mg/kg, 3mg/kg to 7 mg/kg, 4 mg/kg to 6 mg/kg, and the like.

In some embodiments, the compositions are administered at a dosage sothat the agent has an in vivo concentration of less than 500 nM, lessthan 400 nM, less than 300 nM, less than 250 nM, less than 200 nM, lessthan 150 nM, less than 100 nM, less than 50 nM, less than 25 nM, lessthan 20, nM, less than 10 nM, less than 5 nM, less than 1 nM, less than0.5 nM, less than 0.1 nM, less than 0.05, less than 0.01, nM, less than0.005 nM, less than 0.001 nM after 15 mins, 30 mins, 1 hr, 1.5 hrs, 2hrs, 2.5 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs,11 hrs, 12 hrs or more of time of administration.

With respect to duration and frequency of treatment, it is typical forskilled clinicians to monitor subjects in order to determine when thetreatment is providing therapeutic benefit, and to determine whether toincrease or decrease dosage, increase or decrease administrationfrequency, discontinue treatment, resume treatment or make otheralteration to treatment regimen. The dosing schedule can vary from oncea week to daily depending on a number of clinical factors, such as thesubject's sensitivity to the polypeptides. The desired dose can beadministered everyday or every third, fourth, fifth, or sixth day. Thedesired dose can be administered at one time or divided into subdoses,e.g., 2-4 subdoses and administered over a period of time, e.g., atappropriate intervals through the day or other appropriate schedule.Such sub-doses can be administered as unit dosage forms. In someembodiments of the aspects described herein, administration is chronic,e.g., one or more doses daily over a period of weeks or months. Examplesof dosing schedules are administration daily, twice daily, three timesdaily or four or more times daily over a period of 1 week, 2 weeks, 3weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6months or more.

In some embodiments, the agent (e.g., a nucleic acid agent or anexpression vector) can be formulated as a liposome. There are manyorganized surfactant structures besides microemulsions that have beenstudied and used for the formulation of drugs. These include monolayers,micelles, bilayers and vesicles. Vesicles, such as liposomes, haveattracted great interest because of their specificity and the durationof action they offer from the standpoint of drug delivery. As used inthe present invention, the term “liposome” means a vesicle composed ofamphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to traverse intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent can act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4(6):466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside GM1 or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C_(1215G), thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describes PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes can include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

In some embodiments, the agent (e.g., a nucleic acid agent or anexpression vector) can be fully encapsulated in the lipid formulation,e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipidparticle. As used herein, the term “SNALP” refers to a stable nucleicacid-lipid particle, including SPLP. As used herein, the term “SPLP”refers to a nucleic acid-lipid particle comprising plasmid DNAencapsulated within a lipid vesicle. SNALPs and SPLPs typically containa cationic lipid, a non-cationic lipid, and a lipid that preventsaggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs andSPLPs are extremely useful for systemic applications, as they exhibitextended circulation lifetimes following intravenous (i.v.) injectionand accumulate at distal sites (e.g., sites physically separated fromthe administration site). SPLPs include “pSPLP,” which include anencapsulated condensing agent-nucleic acid complex as set forth in PCTPublication No. WO 00/03683. The particles of the present inventiontypically have a mean diameter of about 50 nm to about 150 nm, moretypically about 60 nm to about 130 nm, more typically about 70 nm toabout 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCTPublication No. WO 96/40964. For SNALP formulation, the particle size isat least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm,and at least 120 nm. The suitable range is typically about at least 50nm to about at least 110 nm, about at least 60 nm to about at least 100nm, or about at least 80 nm to about at least 90 nm.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to nucleic acid) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.C1),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.C1),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech Gi), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

The non-cationic lipid can be an anionic lipid or a neutral lipidincluding, but not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the agents (e.g. nucleic acid agents or expressionvectors) can be prepared and formulated as emulsions. Emulsions aretypically heterogeneous systems of one liquid dispersed in another inthe form of droplets usually exceeding 0.1 μm in diameter (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials is also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

In one embodiment the agents can be formulated as microemulsions. Amicroemulsion can be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and DrugDelivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typicallymicroemulsions are systems that are prepared by first dispersing an oilin an aqueous surfactant solution and then adding a sufficient amount ofa fourth component, generally an intermediate chain-length alcohol toform a transparent system. Therefore, microemulsions have also beendescribed as thermodynamically stable, isotropically clear dispersionsof two immiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

As used herein, the term “administer” refers to the placement of acomposition into a subject by a method or route which results in atleast partial localization of the composition at a desired site suchthat desired effect is produced. A compound or composition describedherein can be administered by any appropriate route known in the artincluding, but not limited to, oral or parenteral routes, includingintravenous, intramuscular, subcutaneous, transdermal, airway (aerosol),pulmonary, nasal, rectal, and topical (including buccal and sublingual)administration.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. Inpreferred embodiments, the compositions are administered by intravenousinfusion or injection. In some embodiments, the compound is administereddirectly into the central nervous system.

By “treatment”, “prevention” or “amelioration” of a disease or disorderis meant delaying or preventing the onset of such a disease or disorder,reversing, alleviating, ameliorating, inhibiting, slowing down orstopping the progression, aggravation or deterioration the progressionor severity of a condition associated with such a disease or disorder.In one embodiment, at least one symptom of a disease or disorder isalleviated by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, or at least 50%.

The term “treatment”, with respect to treatment of Alzheimer's diseaseor a disease associated with Aβ accumulation or aggregation refers to,inter alia, preventing the development of the disease, or altering thecourse of the disease (for example, but not limited to, slowing theprogression of the disease), or reversing a symptom of the disease orreducing one or more symptoms and/or one or more biochemical markers ina subject, preventing one or more symptoms from worsening orprogressing, promoting recovery or improving prognosis, and/orpreventing disease in a subject who is free therefrom as well as slowingor reducing progression of existing disease. For a given subject,improvement in a symptom, its worsening, regression, or progression canbe determined by an objective or subjective measure. Modification of oneor more biochemical markers or presence of beta amyloid in the CSF forexample can be measured. For example, but not limited to, a reduction ina biochemical marker of Alzheimer's disease, for example a reduction inamyloid plaque deposition by 10%, or a reduction in the activation ofglial cells, for example a reduction in cells expressing GFAP by 10%,would be considered effective treatments by the methods as disclosedherein. As alternative examples, a reduction in a symptom, for example,a slowing of the rate of memory loss by 10% or a cessation of the ratememory decline, or a reduction in memory loss by 10% or an improvementin memory by 10% would also be considered as affective treatments by themethods as disclosed herein.

Further, as used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of Alzheimer'sdisease. Beneficial or desired clinical results can include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. Any particular treatment regimen can provide one or moresuch clinical results in one or more patients, and need not provide allsuch clinical results. “Treatment” can also mean prolonging survival ascompared to expected survival if not receiving treatment. Those in needof treatment include those already with the condition or disorder aswell as those prone to have the condition or disorder or those in whichthe condition or disorder is to be prevented.

As used herein, the terms “effective” and “effectiveness” includes bothpharmacological effectiveness and physiological safety. Pharmacologicaleffectiveness refers to the ability of the treatment to result in adesired biological effect in the patient. Physiological safety refers tothe level of toxicity, or other adverse physiological effects at thecellular, organ and/or organism level (often referred to asside-effects) resulting from administration of the treatment. “Lesseffective” means that the treatment results in a therapeuticallysignificant lower level of pharmacological effectiveness and/or atherapeutically greater level of adverse physiological effects.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments, the subject is a mammal,e.g., a primate, e.g., a human. The terms, “patient” and “subject” areused interchangeably herein. The terms, “patient” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models ofneuro-inflammatory disorders.

In addition, the methods described herein can be used to treatdomesticated animals and/or pets. A subject can be male or female. Asubject can be one who has been previously diagnosed with or identifiedas suffering from or having neuro-inflammatory disease or disorder, butneed not have already undergone treatment.

Alzheimer's Disease

Alzheimer's disease (AD) is a progressive disease resulting in seniledementia. See generally Selkoe, TINS 16, 403-409 (1993); Hardy et al.,WO 92/13069; Selkoe, J. Neuropathol. Exp. Neurol. 53, 438-447 (1994);Duff et al., Nature 373, 476-477 (1995); Games et al., Nature 373, 523(1995). Broadly speaking the disease falls into two categories: lateonset, which occurs in old age (65+ years) and early onset, whichdevelops well before the senile period, i.e, between 35 and 60 years. Inboth types of disease, the pathology is the same but the β abnormalitiestend to be more severe and widespread in cases beginning at an earlierage. The disease is characterized at the macroscopic level bysignificant brain shrinkage away from the cranial vault as seen in MRIimages as a direct result of neuronal loss and by two types ofmacroscopic lesions in the brain, senile plaques and neurofibrillarytangles. Senile plaques are areas comprising disorganized neuronalprocesses up to 150 μm across and extracellular amyloid deposits, whichare typically concentrated at the center and visible by microscopicanalysis of sections of brain tissue. Neurofibrillary tangles areintracellular deposits of tau protein consisting of two filamentstwisted about each other in pairs.

The principal constituent of the plaques is a peptide termed Aβ orβ-amyloid peptide. Aβ peptide is an internal fragment of 39-43 aminoacids of a precursor protein termed amyloid precursor protein (APP).Several mutations within the APP protein have been correlated with thepresence of Alzheimer's disease. See, e.g., Goate et al., Nature 349,704) (1991) (valine⁷¹⁷ to isoleucine); Chartier Harlan et al. Nature353, 844 (1991)) (valine⁷¹⁷ to glycine); Murrell et al., Science 254, 97(1991) (valine⁷¹⁷ to phenylalanine); Mullan et al., glycine); Murrell etal., Science 254, 97 (1991) (valine⁷¹⁷ to phenylalanine); Mullan et al.,Nature Genet. 1, 345 (1992) (a double mutation changinglysine⁵⁹⁵-methionine⁵⁹⁶ to asparagine⁵⁹⁵-leucine⁵⁹⁶). Such mutations arethought to cause Alzheimer's disease by increased or altered processingof APP to Aβ, particularly processing of APP to increased amounts of thelong form of Aβ (i.e., Aβ1-42 and Aβ1-43). Mutations in other genes,such as the presenilin genes, PS1 and PS2, are thought indirectly toaffect processing of APP to generate increased amounts of long form Aβ(see Hardy, TINS 20, 154 (1997)). These observations indicate that Aβ,and particularly its long form, is a causative element in Alzheimer'sdisease.

Aβ, also known as β-amyloid peptide, or A4 peptide (see U.S. Pat. No.4,666,829; Glenner & Wong, Biochem. Biophys. Res. Commun. 120, 1131(1984)), is a peptide of 39-43 amino acids, is the principal componentof characteristic plaques of Alzheimer's disease. Aβ is generated byprocessing of a larger protein APP by two enzymes, termed β and γsecretases (see Hardy, TINS 20, 154 (1997)). Known mutations in APPassociated with Alzheimer's disease occur proximate to the site of β orγ-secretase, or within Aβ. For example, position 717 is proximate to thesite of γ-secretase cleavage of APP in its processing to Aβ, andpositions 670/671 are proximate to the site of β-secretase cleavage. Itis believed that the mutations cause AD disease by interacting with thecleavage reactions by which Aβ is formed so as to increase the amount ofthe 42/43 amino acid form of Aβ generated.

Aβ has the unusual property that it can fix and activate both classicaland alternate complement cascades. In particular, it binds to C1q andultimately to C3bi. This association facilitates binding to macrophagesleading to activation of B cells. In addition, C3bi breaks down furtherand then binds to CR2 on B cells in a T cell dependent manner leading toa 10,000 increase in activation of these cells. This mechanism causes Aβto generate an immune response in excess of that of other antigens.

Most therapeutic strategies for Alzheimer's disease are aimed atreducing or eliminating the deposition of Aβ42 in the brain, typicallyvia reduction in the generation of Aβ42 from APP and/or some means oflowering existing Aβ42 levels from sources that directly contribute tothe deposition of this peptide in the brain (De Felice and Ferreira,2002). A partial list of aging-associated causative factors in thedevelopment of sporadic Alzheimer's disease includes a shift in thebalance between Aβ peptide production and its clearance from neuronsthat favors intracellular accumulation, increased secretion of Aβpeptides by neurons into the surrounding extracellular space, increasedlevels of oxidative damage to these cells, and global brainhypoperfusion and the associated compensatory metabolic shifts inaffected.

The Aβ42 that deposits within neurons and plaques could also originatefrom outside of the neurons (exogenous Aβ42) during Alzheimer's diseasepathogenesis. Levels of soluble Aβ peptides in the blood are known to bemuch higher than in the interstitial space and CSF in the brains ofhealthy individuals with blood as a source of exogenous Aβ peptides thateventually deposit in the Alzheimer's disease brain. However, except fortrace amounts of Aβ that are actively transported across endothelialcells, it is well-known that access of blood-borne Aβ peptides to braintissue in normal healthy individuals is effectively blocked by theintegrity of the blood-brain barrier (BBB).

Genetic markers of risk toward Alzheimer's disease include mutations inthe APP gene, particularly mutations at position 717 and positions 670and 671 referred to as the Hardy and Swedish mutations respectively (seeHardy, TINS, supra). Other markers of risk are mutations in thepresenilin genes, PS1 and PS2, and ApoE4, family history of Alzheimer'sdisease, hypercholesterolemia or atherosclerosis. Subjects presentlysuffering from Alzheimer's disease can be recognized from characteristicdementia, as well as the presence of risk factors described above. Inaddition, a number of diagnostic tests are available for identifyingsubjects who have Alzheimer's disease. These include measurement of CSFtau and Aβ42 levels. Elevated tau and increased Aβ42 levels signify thepresence of Alzheimer's disease. Individuals suffering from Alzheimer'sdisease can also be diagnosed by MMSE or ADRDA criteria. The tissuesample for analysis is typically blood, plasma, serum, mucus or cerebralspinal fluid from the patient. The sample is analyzed for indicia of animmune response to any forms of Aβ peptide, typically Aβ42. The immuneresponse can be determined from the presence of, e.g., antibodies orT-cells that specifically bind to Aβ peptide. ELISA methods of detectingantibodies specific to Aβ are described in the Examples section.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically entails multipledosages over a period of time.

Methods to Identify Subjects for Risk of or Having Alzheimer's Disease.

Subjects amenable to treatment using the methods as disclosed hereininclude subjects at risk of a neurodegenerative disease, for exampleAlzheimer's Disease but not showing symptoms, as well as subjectsshowing symptoms of the neurodegenerative disease, for example subjectswith symptoms of Alzheimer's Disease. Subjects can be screened for theirlikelihood of having or developing Alzheimer's Disease based on a numberof biochemical and genetic markers.

One can also diagnose a subject with increased risk of developingAlzheimer's Disease using genetic markers for Alzheimer's Disease.Genetic abnormality in a few families has been traced to chromosome 21(St. George-Hyslop et al., Science 235:885-890, 1987). One geneticmarker is, for example mutations in the APP gene, particularly mutationsat position 717 and positions 670 and 671 referred to as the Hardy andSwedish mutations respectively (see Hardy, TINS, supra). Other markersof risk are mutations in the presenilin genes, PS1 and PS2, and ApoE4,family history of Alzheimer's Disease, hypercholesterolemia oratherosclerosis. Subjects with APP, PS1 or PS2 mutations are highlylikely to develop Alzheimer's disease. ApoE is a susceptibility gene,and subjects with the e4 isoform of ApoE (ApoE4 isoform) have anincreased risk of developing Alzheimer's disease. Test for subjects withApoE4 isoform are disclosed in U.S. Pat. No. 6,027,896, which isincorporated in its entirety herein by reference. Other genetic linkshave been associated with increased risk of Alzheimer's disease, forexample variances in the neuronal sortilin-related receptor SORL1 mayhave increased likelihood of developing late-onset Alzheimer's disease(Rogaeva at al., Nat Genet. 2007 February; 39(2):168-77). Otherpotential Alzheimer disease susceptibility genes, include, for exampleACE, CHRNB2, CST3, ESR1, GAPDHS, IDE, MTHFR, NCSTN, PRNP, PSEN1, TF,TFAM and TNF and be used to identify subjects with increased risk ofdeveloping Alzheimer's disease (Bertram et al, Nat Genet. 2007 January;39(1):17-23), as well as variances in the alpha-T catenin (VR22) gene(Bertram et al, J Med Genet. 2007 January; 44(1):e63) andInsulin-degrading enzyme (IDE) and Kim et al, J Biol Chem. 2007;282:7825-32). As disclosed in the present disclosure, CD33 protein orgene encoding the same is also associated with Alzheimer's disease.

One can also diagnose a subject with increased risk of developingAlzheimer's disease on the basis of a simple eye test, where thepresence of cataracts and/or Abeta in the lens identifies a subject withincreased risk of developing Alzheimer's Disease. Methods to detectAlzheimer's disease include using a quasi-elastic light scatteringdevice (Goldstein et al., Lancet. 2003; 12; 361:1258-65) from Neuroptix,using Quasi-Elastic Light Scattering (QLS) and Fluorescent LigandScanning (FLS) and a Neuroptix™ QEL scanning device, to enablenon-invasive quantitative measurements of amyloid aggregates in the eye,to examine and measure deposits in specific areas of the lens as anearly diagnostic for Alzheimer's disease. Method to diagnose a subjectat risk of developing Alzheimer's disease using such a method ofnon-invasive eye test are disclosed in U.S. Pat. No. 7,107,092, which isincorporated in its entirety herein by reference.

Individuals presently suffering from Alzheimer's disease can berecognized from characteristic dementia, as well as the presence of riskfactors described above. In addition, a number of diagnostic tests areavailable for identifying individuals who have AD. These includemeasurement of CSF tau and Ax3b242 levels. Elevated tau and decreasedAx3b242 levels signify the presence of Alzheimer's Disease.

There are two alternative “criteria” which are utilized to clinicallydiagnose Alzheimer's Disease: the DSM-IIIR criteria and the NINCDS-ADRDAcriteria (which is an acronym for National Institute of Neurological andCommunicative Disorders and Stroke (NINCDS) and the Alzheimer's Diseaseand Related Disorders Association (ADRDA); see McKhann et al., Neurology34:939-944, 1984). Briefly, the criteria for diagnosis of Alzheimer'sDisease under DSM-IIIR include (1) dementia, (2) insidious onset with agenerally progressive deteriorating course, and (3) exclusion of allother specific causes of dementia by history, physical examination, andlaboratory tests. Within the context of the DSM-IIIR criteria, dementiais understood to involve “a multifaceted loss of intellectual abilities,such as memory, judgement, abstract thought, and other higher corticalfunctions, and changes in personality and behaviour.” (DSM-1IR, 1987).

In contrast, the NINCDS-ADRDA criteria sets forth three categories ofAlzheimer's Disease, including “probable,” “possible,” and “definite”Alzheimer's Disease. Clinical diagnosis of “possible” Alzheimer'sDisease may be made on the basis of a dementia syndrome, in the absenceof other neurologic, psychiatric or systemic disorders sufficient tocause dementia. Criteria for the clinical diagnosis of “probable”Alzheimer's Disease include (a) dementia established by clinicalexamination and documented by a test such as the Mini-Mental test(Foldstein et al., J. Psych. Res. 12:189-198, 1975); (b) deficits in twoor more areas of cognition; (c) progressive worsening of memory andother cognitive functions; (d) no disturbance of consciousness; (e)onset between ages 40 and 90, most often after age 65; and (f) absenceof systemic orders or other brain diseases that could account for thedementia. The criteria for definite diagnosis of Alzheimer's Diseaseinclude histopathologic evidence obtained from a biopsy, or afterautopsy. Since confirmation of definite Alzheimer's Disease requireshistological examination from a brain biopsy specimen (which is oftendifficult to obtain), it is rarely used for early diagnosis ofAlzheimer's Disease.

One can also use neuropathologic diagnosis of Alzheimer's Disease, wherethe numbers of plaques and tangles in the neurocortex (frontal,temporal, and parietal lobes), hippocampus and amygdala are analyzed(Khachaturian, Arch. Neurol. 42:1097-1105; Esiri, “Anatomical Criteriafor the Biopsy diagnosis of Alzheimer's Disease,” Alzheimer's Disease,Current Research in Early Diagnosis, Becker and Giacobini (eds.), pp.239-252, 1990).

One can also use quantitative electroencephalographic analysis (EEG) todiagnose Alzheimer's Disease. This method employs Fourier analysis ofthe beta, alpha, theta, and delta bands (Riekkinen et al., “EEG in theDiagnosis of Early Alzheimer's Disease,” Alzheimer's Disease, CurrentResearch in Early Diagnosis, Becker and Giacobini (eds.), pp. 159-167,1990) for diagnosis of Alzheimer's Disease.

One can also diagnose Alzheimer's Disease by quantifying the degree ofneural atrophy, since such atrophy is generally accepted as aconsequence of Alzheimer's Disease. Examples of these methods includecomputed tomographic scanning (CT), and magnetic resonance imaging (MRI)(Leedom and Miller, “CT, MRI, and NMR Spectroscopy in Alzheimer'sDisease,” Alzheimer's Disease, Current Research in Early Diagnosis,Becker and Giacobini (eds.), pp. 297-313, 1990).

One can also diagnose Alzheimer's Disease by assessing decreasedcerebral blood flow or metabolism in the posterior temporoparietalcerebral cortex by measuring decreased blood flow or metabolism bypositron emission tomography (PET) (Parks and Becker, “Positron EmissionTomography and Neuropsychological Studies in Dementia,” Alzheimer'sDisease's, Current Research in Early Diagnosis, Becker and Giacobini(eds.), pp. 315-327, 1990), single photon emission computed tomography(SPECT) (Mena et al., “SPECT Studies in Alzheimer's Type DementiaPatients,” Alzheimer's Disease, Current Research in Early Diagnosis,Becker and Giacobini (eds.), pp. 339-355, 1990), and xenon inhalationmethods (Jagust et al., Neurology 38:909-912; Prohovnik et al.,Neurology 38:931-937; and Waldemar et al., Senile Dementias: IIInternational Symposium, pp. 399407, 1988).

One can also immunologically diagnose Alzheimer's disease (Wolozin,“Immunochemical Approaches to the Diagnosis of Alzheimer's Disease,”Alzheimer's Disease, Current Research in Early Diagnosis, Becker andGiacobini (eds.), pp. 217-235, 1990). Wolozin and coworkers (Wolozin etal., Science 232:648-650, 1986) produced a monoclonal antibody “Alz50,”that reacts with a 68-kDa protein “A68,” which is expressed in theplaques and neuron tangles of patients with Alzheimer's disease. Usingthe antibody Alz50 and Western blot analysis, A68 was detected in thecerebral spinal fluid (CSF) of some Alzheimer's patients and not in theCSF of normal elderly patients (Wolozin and Davies, Ann. Neurol.22:521-526, 1987).

One can also diagnose Alzheimer's disease using neurochemical markers ofAlzheimer's disease. Neurochemical markers which have been associatedwith Alzheimer's Disease include reduced levels of acetylcholinesterase(Giacobini and Sugaya, “Markers of Cholinergic Dysfunction inAlzheimer's Disease,” Alzheimer's Disease, Current Research in EarlyDiagnosis, Becker and Giacobini (eds.), pp. 137-156, 1990), reducedsomatostatin (Tamminga et al., Neurology 37:161-165, 1987), a negativerelation between serotonin and 5-hydroxyindoleacetic acid (Volicer etal., Arch Neurol. 42:127-129, 1985), greater probenecid-induced rise inhomovanyllic acid (Gibson et al., Arch. Neurol. 42:489-492, 1985) andreduced neuron-specific enolase (Cutler et al., Arch. Neurol.43:153-154, 1986).

Other methods to diagnose a patient at risk of or having aneurodegenerative disease or disorder, such as Alzheimer's Diseaseincludes measurement of CD33 activity and/or expression using themethods as disclosed herein, for example using quantitative RT-PCR asdescribed in the examples section.

Embodiments of the various aspects disclosed herein can be described byone or more of the following numbered paragraphs:

-   -   1. A method of treating a neuro-inflammation disorder in a        subject, the method comprising administering to a subject in        need thereof an effective amount of an agent that inhibits or        reduces the expression or activity of CD33 protein.    -   2. The method of paragraph 1, wherein said agent is selected        from the group consisting of small or large organic or inorganic        molecules, nucleic acids, nucleic acid analogs and derivatives,        peptides, peptidomimetics, proteins, antibodies and antigen        binding fragments thereof, monosaccharides, disaccharides,        trisaccharides, oligosaccharides, polysaccharides, lipids,        glycosaminoglycans, an extract made from biological materials,        and any combinations thereof    -   3. The method of paragraph 1 or 2, wherein the agent is a        nucleic acid selected from the group consisting of siRNA, shRNA,        miRNA, anti-microRNA, antisense RNA or oligonucleotide, aptamer,        ribozyme, and any combinations thereof.    -   4. The method of any of paragraphs 1-3, wherein the agent is an        RNAi agent.    -   5. The method of any of paragraphs 1-4, wherein the agent is a        nucleic acid and the method comprises administering a vector        encoding/expressing the agent to the subject.    -   6. The method of paragraph 5, wherein the vector is a viral        vector    -   7. The method of paragraph 6, wherein the viral vector is an        adeno-associated virus (AAV) vector.    -   8. The method of paragraph 1 or 2, wherein the agent is a small        molecule selected from the group consisting of sialic acid        analogues and derivatives.    -   9. The method of paragraph 1 or 2, wherein the agent is a        monoclonal antibody.    -   10. The method of paragraph 1, 2 or 9, wherein the agent is a        humanized antibody.    -   11. The method of paragraph 1, 2, 9, or 10, wherein the agent is        an anti-CD33 antibody or an antigen binding fragment thereof.    -   12. The method of any of paragraphs 1-11, wherein the agent        crosses or is formulated to cross the blood-brain barrier.    -   13. A method of treating a neuro-inflammation disorder in a        subject, the method comprising administering to a subject in        need thereof a nucleic acid encoding a CD33 protein, wherein the        CD33 protein lacks sialic acid binding domain.    -   14. The method of paragraph 13, wherein the CD33 protein        comprises an amino acid sequence SEQ ID NO: 8.    -   15. The method of paragraph 13 or 14, wherein the nucleic acid        is a vector.    -   16. The method of paragraph 15, wherein the vector is a viral        vector    -   17. The method of paragraph 16, wherein the viral vector is an        AAV vector.    -   18. The method of paragraph 13 or 14, wherein the nucleic acid        is modified RNA.    -   19. The method of any of paragraphs 1-18, wherein the        neuro-inflammation disorder is a neurodegenerative disease or        disorder.    -   20. The method of paragraph 19, wherein the neurodegenerative        disease or disorder is selected from the group consisting of        Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's        Disease, Amyotrophic Lateral Sclerosis (ALS, also termed Lou        Gehrig's disease) and Multiple Sclerosis (MS).    -   21. The method of paragraph 20, wherein the neurodegenerative        disease or disorder is Alzheimer's Disease.    -   22. The method of any of paragraphs 1-21, wherein the subject is        mammalian.    -   23. The method of any of paragraph 22, wherein the subject is        human.    -   24. The method of any of paragraphs 1-23, wherein the agent        decreases beta amyloid accumulation in the brain of the subject.    -   25. The method of paragraph 24, wherein the beta amyloid is        Aβ-42    -   26. A method of decreasing beta amyloid accumulation in the        brain of a subject, the method comprising administering to a        subject in need thereof an effective amount of an agent that        inhibits or reduces the expression or activity of CD33 protein.    -   27. The method of paragraph 26, wherein said agent is selected        from the group consisting of small or large organic or inorganic        molecules, nucleic acids, nucleic acid analogs and derivatives,        peptides, peptidomimetics, proteins, antibodies and antigen        binding fragments thereof, monosaccharides, disaccharides,        trisaccharides, oligosaccharides, polysaccharides, lipids,        glycosaminoglycans, an extract made from biological materials,        and any combinations thereof    -   28. The method of paragraph 26 or 27, wherein the agent is a        nucleic acid selected from the group consisting of siRNA, shRNA,        miRNA, anti-microRNA, antisense RNA, aptamer, ribozyme, and any        combinations thereof    -   29. The method of any of paragraphs 26-28, wherein the agent is        an RNAi agent.    -   30. The method of any of paragraphs 26-29, wherein the agent is        a nucleic acid and the method comprises administering a vector        encoding/expressing the agent to the subject.    -   31. The method of paragraph 30, wherein the vector is a viral        vector    -   32. The method of paragraph 31, wherein the viral vector is an        adeno-associated virus (AAV) vector.    -   33. The method of paragraph 26 or 27, wherein the agent is a        small molecule selected from the group consisting of sialic acid        analogues and derivatives.    -   34. The method of paragraph 26 or 27, wherein the agent is a        monoclonal antibody.    -   35. The method of paragraph 26, 37 or 34, wherein the agent is a        humanized antibody.    -   36. The method of paragraph 26, 27, 34, or 35, wherein the agent        is an anti-CD33 antibody or an antigen binding fragment thereof.    -   37. The method of any of paragraphs 26-36, wherein the agent        crosses the blood-brain barrier.    -   38. A method of decreasing beta amyloid accumulation in the        brain of a subject, the method comprising administering to a        subject in need thereof a vector expressing administering to a        subject in need thereof a nucleic acid encoding a CD33 protein,        wherein the CD33 protein lacks sialic acid binding domain.    -   39. The method of paragraph 38, wherein the CD33 protein        comprises an amino acid sequence SEQ ID NO: 8.    -   40. The method of paragraph 38 or 39, wherein the nucleic acid        is a vector.    -   41. The method of paragraph 40, wherein the vector is a viral        vector    -   42. The method of paragraph 41, wherein the viral vector is an        adenovirus vector.    -   43. The method of paragraph 38 or 39, wherein the nucleic acid        is modified RNA.    -   44. The method of any of paragraphs 26-43, wherein the subject        is in need for treating a neuro-inflammation disorder.    -   45. The method of paragraph 44, wherein the neuro-inflammation        disorder is a neurodegenerative disease or disorder.    -   46. The method of paragraph 45, wherein the neurodegenerative        disease or disorder is selected from the group consisting of        Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's        Disease, Amyotrophic Lateral Sclerosis (ALS, also termed Lou        Gehrig's disease) and Multiple Sclerosis (MS).    -   47. The method of paragraph 46, wherein the neurodegenerative        disease or disorder is Alzheimer's Disease.    -   48. The method of any of paragraphs 26-47, wherein the subject        is mammalian.    -   49. The method of any of paragraph 48, wherein the subject is        human.    -   50. The method of any of paragraphs 26-49, wherein the beta        amyloid is Aβ-42.    -   51. The use of an agent which inhibits or reduces the expression        or activity of CD33 protein for the preparation of a medicament        for treatment or prevention of a neuro-inflammatory disorder.    -   52. The use of a vector which expresses a CD33 protein lacking        sialic acid binding domain for the preparation of a medicament        for treatment or prevention of a neuro-inflammatory disorder.

Some Selected Definitions

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected herein. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the art to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±5% of the value being referred to. For example, about 100 meansfrom 95 to 105.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” The abbreviation, “e.g.” is derived from the Latinexempli gratia, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”used herein in context of CD33 expression and/or activity means that theexpression or activity of CD33 protein or variants or homologues thereofis reduced to an extent, and/or for a time, sufficient to produce thedesired effect.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means at least two standarddeviation (2SD) away from a reference level. The term refers tostatistical evidence that there is a difference. It is defined as theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true.

The term “disease” or “disorder” is used interchangeably herein, andrefers to any alteration in state of the body or of some of the organs,interrupting or disturbing the performance of the functions and/orcausing symptoms such as discomfort, dysfunction, distress, or evendeath to the person afflicted or those in contact with a person. Adisease or disorder can also relate to a distemper, ailing, ailment,malady, disorder, sickness, illness, complaint, inderdisposion oraffectation.

As used herein, “gene silencing” or “gene silenced” in reference to anactivity of n RNAi molecule, for example a siRNA or miRNA refers to adecrease in the mRNA level in a cell for a target gene by at least about5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of themRNA level found in the cell without the presence of the miRNA or RNAinterference molecule. In one preferred embodiment, the mRNA levels aredecreased by at least about 70%, about 80%, about 90%, about 95%, about99%, about 100%.

The term “analog” as used herein refers to a compound that results fromsubstitution, replacement or deletion of various organic groups orhydrogen atoms from a parent compound. An analog is structurally similarto the parent compound, but can differ by even a single element of thesame valence and group of the periodic table as the element it replaces.

The term “derivative” as used herein refers to a chemical substancerelated structurally to another, i.e., an “original” substance, whichcan be referred to as a “parent” compound. A “derivative” can be madefrom the structurally-related parent compound in one or more steps. Thephrase “closely related derivative” means a derivative whose molecularweight does not exceed the weight of the parent compound by more than50%. The general physical and chemical properties of a closely relatedderivative are also similar to the parent compound.

To the extent not already indicated, it will be understood by those ofordinary skill in the art that any one of the various embodiments hereindescribed and illustrated can be further modified to incorporatefeatures shown in any of the other embodiments disclosed herein.

EXAMPLES

The following examples illustrate some embodiments and aspects of theinvention. It will be apparent to those skilled in the relevant art thatvarious modifications, additions, substitutions, and the like can beperformed without altering the spirit or scope of the invention, andsuch modifications and variations are encompassed within the scope ofthe invention as defined in the claims which follow. The followingexamples do not in any way limit the invention.

Example 1 Alzheimer's Disease Risk Gene CD33 Inhibits Microglial Uptakeof Amyloid Beta

The transmembrane protein CD33 is a sialic acid-bindingimmunoglobulin-like lectin that regulates innate immunity but has noknown functions in the brain. It was previously shown that the CD33 geneis a risk factor for Alzheimer's disease (AD). Here, increasedexpression of CD33 in microglial cells in AD brain was observed. Theminor allele of the CD33 single nucleotide polymorphism rs3865444, whichconfers protection against AD, was associated with reductions in bothCD33 expression and insoluble amyloid beta 42 (Aβ42) levels in AD brain.Furthermore, the numbers of CD33-immunoreactive microglia werepositively correlated with insoluble Aβ42 levels and plaque burden in ADbrain. CD33 inhibited uptake and clearance of Aβ42 in microglial cellcultures. Finally, brain levels of insoluble Aβ42 as well as amyloidplaque burden were markedly reduced in APP_(Swe)/PS1_(ΔE9)/CD33^(−/−)mice. Therefore, CD33 inactivation mitigates Aβ pathology and CD33inhibition represents a novel therapy for AD.

Here, CD33 is shown to be expressed in microglial cells in the humanbrain. CD33 protein levels as well as the number of CD33-positivemicroglia are increased in AD brains relative to age-matched controls.Conversely, it is shown that the minor allele of the CD33 SNP rs3865444,which protects against AD, leads to reductions in both CD33 microglialexpression and levels of insoluble Aβ42 in AD brain. Furthermore, thenumbers of CD33-immunoreactive microglia positively correlate withinsoluble Aβ42 levels and the amyloid plaque burden in AD cases. Usingcultured primary and BV2 microglial cells, CD33 is shown to be bothrequired and sufficient to inhibit the microglial uptake of Aβ42, thusimpairing Aβ42 clearance. Finally, APP_(Swe)/PS1_(ΔE9) transgenic micein which the CD33 gene was knocked out exhibited a marked reduction ofinsoluble Aβ42 levels and Aβ plaque burden, indicating that CD33promotes the Aβ42 pathology in vivo. Collectively, these results suggestthat CD33 activity in microglia promotes Aβ42 pathology in AD. They alsoraise the possibility that the loss of microglial degradative capacityof Aβ in AD could be reversed therapeutically by inhibition of CD33activity.

Increased CD33 Expression in AD.

To assess the role of CD33 in AD pathology, the expression of CD33 wasinitially assessed in post-mortem brain samples from 25 AD patients and15 age-matched non-demented controls (cohort characteristics in Table1). To investigate the relationship between CD33 mRNA levels and AD,quantitative RT-PCR was performed on total mRNA extracted from frozencortical samples. This revealed a five-fold increase in CD33 mRNA levelsin AD cases relative to controls (FIG. 1A, p<0.01, student's t-test).Normalization of CD33 mRNA levels using GAPDH and β-Actin mRNAs led tosimilar results (FIG. 1A). Next, it was assessed whether CD33 proteinlevels are increased in the frontal cortex in AD. Western blotting usinga CD33-specific antibody (Hoyer et al., 2008; Rollins-Raval and Roth,2012) revealed a two-fold increase in CD33 protein levels in AD samplesrelative to controls (FIGS. 1B and 1C, p<0.01, student's t-test). Thissignificant increase was also observed when normalizing CD33 levels tothe levels of the microglial marker Iba1 (FIG. 1C, p<0.05, student'st-test), indicating that this difference is not explained by anincreased number of microglia in AD. Using stereology-based quantitativemethods, it was previously observed that the total number ofIba1-positive microglia is not significantly different between AD andnon-demented subjects (unpublished data). Thus, CD33 mRNA and proteinlevels are increased in AD brain.

TABLE 1 Characteristics of AD cases and controls used in the studyCharacteristics Controls (n = 15) AD (n = 25) Age at death (years) 79.9± 11.2 79.2 ± 8.3 Disease duration (years) NA 10.96 Males/Females40%/60% 28%/72% APOEε4 carriers 5 (33.33%) 18 (72%) APOEε4 homozygous 08 (32%) carriers Post-mortem interval (hours) 29 ± 9   17 ± 12

The rs3865444 SNP in the CD33 gene was previously reported to conferprotection against AD (Hollingworth et al., 2011; Naj et al., 2011).Thus, it was investigated whether CD33 expression differed in carriersof the major (G) allele, relative to the carriers of the minorprotective (T) allele. All subjects were genotyped by the Taqman assayusing sequence-specific primers, to differentiate between the alleles(Shen et al., 2009). It was found that the minor protective (T) alleleis not associated with changes in CD33 mRNA levels (FIG. 1E; p>0.05,general linear regression model). Different sets of primers were used toamplify different regions of the CD33 mRNA, with similar results (FIGS.7A and 7B). However, remarkably, carriers of the minor (T) allele hadsignificantly reduced CD33 protein levels (normalized to GAPDH or Iba1protein levels), in both AD and control groups (FIG. 1D; p<0.05,student's t-test and FIG. 7C). It was also found that the protective (T)allele is associated with decreased CD33 protein levels (normalized toGAPDH or Iba1 protein levels) in both control and AD groups (FIG. 1E,p<0.05, general linear regression model). Thus, although the rs3865444SNP is located on chromosome 19 at the 51,727,962 base pair (bp)position, upstream of the 5′ untranslated region of the CD33 gene(51,728,335-51,743,274 bps, forward strand) (Hollingworth et al., 2011;Naj et al., 2011), it does not affect CD33 mRNA stability but somehowinfluences mRNA translation or protein stability. One possibility isthat the rs3865444 SNP is in linkage disequilibrium with anotherfunctional variant(s) located in the coding region. The observations ofincreased CD33 expression in the AD brain and the decreased CD33 proteinlevels in the carriers of the protective allele of the CD33 SNPrs3865444 strongly suggest a role for CD33 in AD pathogenesis.

Microglial Localization of CD33 and Relationship to AD.

CD33 has previously been shown to be expressed in cells of the immuneand hematopoietic cell systems (Crocker et al., 2007). Microglial cellsare responsible for the immune surveillance of the brain and regulatecritical processes relevant to AD pathology, including the uptake andclearance of Aβ (Aguzzi et al., 2013; Prinz et al., 2011). Thus, it wasinvestigated whether CD33 is expressed in microglial cells.Immunolabeling of control and AD frontal cortex sections, using aCD33-specific antibody (Hoyer et al., 2008; Rollins-Raval and Roth,2012) and the microglial marker Iba1 revealed a good co-localizationbetween the two proteins (FIGS. 2A and 2C). CD33 was also expressed inneurons (FIGS. 8D and 8E), but not in astrocytes, oligodendrocytes orendothelial cells (FIGS. 8G-8I).

Next, it was investigated whether the number of CD33-immunoreactivecells differed between AD and control brains. Sections immunolabeled forCD33 and stained with diaminobenzidine (DAB) were subjected tostereology-based quantifications, using previously described protocols(Serrano-Pozo et al., 2011). The total numbers of CD33-positive cellsincreased by 48.9% in the AD frontal cortex relative to age-matchedcontrols (FIG. 8A, n=28 AD cases and 18 controls, p<0.001, student'st-test). The CD33-positive microglia was identified using morphologicalcriteria (FIGS. 2B and 2D). Stereology-based quantification of thenumbers of CD33-immunoreactive microglia revealed a marked increase inthe AD frontal cortex relative to age-matched controls (FIG. 2E, n=28 ADcases and 18 controls, p<0.001, student's t-test). It was alsoinvestigated whether the number of CD33-positive neurons differedbetween AD and control subjects. No significant difference was foundbetween the numbers of CD33-positive neurons in AD and controls (FIGS.8B and 8C). The levels of CD33 protein normalized to Iba1 protein levelspositively correlated with the numbers of CD33-immunoreactive microglialcells, as expected (FIG. 2F, p=0.007, Pearson's correlation test). Tovalidate these findings, the relationship between the levels of CD33protein and those of the microglial marker Iba1 was assessed, usingwestern blotting and frontal cortex protein extracts. A positivecorrelation between CD33 and Iba1 levels was found, both in control(FIG. 2G, p=0.03, Pearson's correlation test) and AD cases (FIG. 2H,p=0.002, Pearson's correlation test).

It was investigated if the minor (T) allele of the CD33 SNP rs3865444 isassociated with changes in the number of CD33-positive microglia.Carriers of the protective (T) allele were found to have lower numbersof CD33-positive microglial cells (FIGS. 2I-2K); this effect wasdose-dependent, i.e. carriers of two (T) alleles exhibited a dramaticreduction of CD33-positive microglia numbers relative to carriers of one(T) allele or carriers of the major (G) allele (FIG. 2L, p<0.01 T/Tversus G/G carriers, one-way Kruskal-Wallis ANOVA, Dunn's test). It wasfound that the minor protective (T) allele is associated with decreasedCD33-immunoreactive microglia numbers in both the control and AD groups(FIG. 2M, p<0.001 and p<0.05 respectively, general linear regressionmodel). Therefore, the numbers of CD33-positive microglia are increasedin AD cases and are reduced in carriers of two protective (T) alleles,suggesting that CD33 activity in microglia might impact the etiologyand/or pathogenesis of AD.

CD33 Microglial Expression Correlates with Amyloid Beta Pathology in AD.

Increased production and deposition of aggregation-prone Aβ species arehallmarks of AD pathology (Selkoe, 2012; Tanzi and Bertram, 2005). Itwas investigated whether Aβ levels were different in carriers of themajor (G) allele of the CD33 SNP rs3865444 in comparison to the carriersof the protective (T) allele. Tris-buffered saline (TBS)-soluble andformic acid (FA)-soluble fractions were generated from the frontalcortex tissue of controls and AD cases (Wang et al., 2011) and thesefractions were used for Aβ ELISA experiments. Remarkably, the carriersof the minor (T) allele were found to have significantly reducedFA-soluble Aβ42 levels but not FA-soluble Aβ40 in comparison to thecarriers of the major (G) allele in AD (FIG. 3A, p<0.01, student'st-test). The minor protective (T) allele was also observed to beassociated with decreased levels of both TBS-soluble Aβ40 and FA-solubleAβ42 in AD cases (FIG. 3B, p<0.05 and p<0.01 respectively, generallinear regression model).

Microglial cells regulate Aβ levels in the brain by a process of uptakeand degradation, which plays a key role in AD pathogenesis (Aguzzi etal., 2013; Prinz et al., 2011). To explore the relationship between CD33microglial expression and amyloid pathology, the AD frontal cortex waslabeled with Thioflavin S (to detect amyloid plaques) and antibodiesdirected against CD33 and the microglial marker Iba1. This revealed abroad distribution of CD33-positive microglia throughout the AD cortextogether with an enrichment of CD33-positive microglia around amyloidplaques (FIGS. 9A-9I). It was then explored the possibility thatincreased CD33 microglial expression in the aging brain promotes Aβpathology by preventing the efficient Aβ clearance. The numbers ofCD33-immunoreactive microglia were found to be positively correlatedwith the levels of FA-soluble Aβ42 in AD brain (FIG. 3C, p=0.02,Spearman's correlation test). It was investigated whether Aβ plaqueburden in AD cases correlates with the numbers of CD33-positivemicroglia. The Aβ plaque burden in the frontal cortex of AD subjects wasestimated as the proportion of area of full-width cortex occupied byAβ-immunoreactive deposits in sections immunostained with an antibodydirected against Aβ (residues 3-7, 10D5) (Serrano-Pozo et al., 2011).Remarkably, a positive correlation between amyloid plaque burden andnumbers of CD33-immunoreactive microglial cells in AD cases was found(FIG. 3D, p=0.017, Spearman's correlation test). These results suggestthat increased microglial expression of CD33 prevents Aβ clearance andstrongly implicate microglial CD33 function in Aβ pathology in AD brain.

CD33 Inactivation Promotes the Uptake of Amyloid Beta by Microglia.

The genetic, biochemical, and histopathological data strongly suggestthat the activity of CD33 in microglia impacts the accumulation of Aβ inthe brain. To test for a causal relationship between CD33 activity andAβ pathology in AD, an in vitro assay of Aβ uptake and clearance wasemployed, using primary microglia isolated from mice with a constitutiveinactivation of the CD33 gene and littermate wild-type (WT) controls.CD33 inactivation in mice does not lead to obvious developmental,histological and behavioral abnormalities, and CD33^(−/−) mice breednormally (Brinkman-Van der Linden et al., 2003). A mixed glial(microglia/astrocyte) primary culture was established using theforebrain of WT and CD33^(−/−) postnatal day 1 (P1) pups as source ofcells, and subsequently enriched for microglial cells (Choi et al.,2008; Gorlovoy et al., 2009); the microglial cultures contained morethan 93% microglial (Iba1-positive) cells. No differences inproliferation, growth and morphological parameters between WT andCD33^(−/−) microglia (data not shown). These cells were stained with aCD33 antibody (FIGS. 10A-10C).

Microglia derived from CD33^(−/−) mice were not immunoreactive to theCD33 antibody, as expected (FIGS. 4A and 4B). The enriched microglialcultures were incubated with Aβ42 for 3 hours, which allows efficientAβ42 uptake. Subsequently, the Aβ42 was washed out and the cells wereincubated for an additional 3 hours, to allow for Aβ42 degradation(Jiang et al., 2008; Mandrekar et al., 2009). To visualize the processof Aβ42 uptake, fluorescently labeled Aβ42 was used (Lee et al., 2012).Visually, there was a strong increase in Aβ42 levels in CD33^(−/−)microglia relative to WT microglia (FIGS. 4A′ and 4B′). Quantificationof the fluorescent Aβ42 signal revealed a significant increase in Aβ42uptake in CD33^(−/−) relative to WT cells (FIG. 4C, p<0.01, student'st-test). The process of Aβ42 degradation cannot be assessed by imagingwith fluorescently labeled Aβ42, because microglial cells degrade Aβ42but they do not completely degrade the attached fluorophore (Mandrekaret al., 2009). To validate the Aβ42 uptake findings, and investigatewhether CD33 inactivation impacts Aβ42 degradation as well, Aβ42 levelswere measured by ELISA on extracts prepared from cultures incubated withunlabeled Aβ42 (FIGS. 4D and 4E). It was found that CD33−/− microgliacontained increased Aβ42 levels after 3 hours of incubation (FIG. 4D),confirming the imaging findings. However, after three hours of Aβ42washout, similar rates of Aβ42 degradation in both CD33^(−/−) and WTmicroglia were found (FIG. 4E). Together, these results suggest thatCD33 directly impacts Aβ42 uptake, but not Aβ42 degradation, inmicroglial cells.

Increased CD33 Levels Inhibit Microglial Uptake of Amyloid Beta.

It was next assessed whether increasing CD33 levels impairs Aβ42 uptakeby microglial cells. For this purpose, the BV2 microglial cell line wasemployed, which was previously found to efficiently take-up and degradeexogenously added Aβ42 (Jiang et al., 2008; Mandrekar et al., 2009).Cells transfected with a WT-CD33 construct displayed a decreased uptakeof fluorescently-labeled Aβ42 relative to cells transfected with anempty construct (FIGS. 5A′ and 5B′ and 5E, p<0.01, one-way ANOVA,Tukey's test). These results were confirmed by ELISA quantifications(FIG. 5F, p<0.05, one-way ANOVA, Tukey's test). Furthermore, ELISA alsorevealed that Aβ42 was degraded at similar rates by cells transfectedwith WT-CD33 or an empty vector (FIG. 5G). Similar differences wereobserved when a construct expressing GFP was used as a control (FIGS.5E-5G). Thus, increasing CD33 levels is sufficient to inhibit Aβ42uptake, but not degradation, by microglial cells.

Cells were next transfected with a mutant version of CD33 in which sevenlysine residues from the intracellular C-terminal domain were mutated toarginine (CD33^(K7R)). This prevents CD33 ubiquitylation and subsequentinternalization from the plasma membrane (Walter et al., 2008). It wasconfirmed that the CD33^(K7R) protein displayed enhanced cell surfaceexpression of CD33 (FIG. 5C) in comparison to the CD33^(WT) protein(FIG. 5B). Expression of the CD33^(K7R) protein led to a furtherinhibition of Aβ42 uptake (FIGS. 5C′ and 5E, p<0.05 CD33^(K7R) versusCD33, one-way ANOVA, Tukey's test and FIG. 5F), but did not impairsubsequent Aβ42 degradation (FIG. 5G), further indicating that CD33inhibits Aβ42 uptake by microglial cells.

CD33 and the related Siglecs perform their biological functions byinteracting with sialic acids, which are attached to the outer membraneof cells, and can mediate cis- or trans-cellular interactions (Paulsonet al., 2012). To determine whether the interaction between CD33 andsialic acids is involved in Aβ42 uptake, a CD33 mutant construct wasemployed in which the sialic acid-binding V-type Immunoglobulin-like(V-Ig) domain was deleted (Perez-Oliva et al., 2011). The mutantCD33ΔV-Ig protein is present at the plasma membrane in BV2 cells, and isexpressed at levels similar to CD33WT (FIGS. 5B and 5D and (Perez-Olivaet al., 2011)). Remarkably, inhibition of Aβ42 uptake by CD33 wascompletely abolished in cells expressing the CD33ΔV-Ig protein; Aβ42levels were similar to those in cells expressing empty vector or GFP(FIGS. 5E and 5F), indicating that sialic acid binding is required forCD33 to mediate Aβ42 uptake. Collectively, these experiments indicatethat CD33 modulates microglial uptake of Aβ42. Specifically, lowerlevels of cell surface CD33 enhance Aβ42 internalization, while higherlevels impair this process. Moreover, interaction of CD33 with sialicacids is necessary to mediate these effects.

CD33 Activity Promotes Amyloid Beta Pathology in Vivo.

The above in vitro experiments, together with the genetic, biochemicaland histopathological observations, strongly implicate CD33 activity inAβ pathology in AD. To provide direct in vivo evidence that CD33activity potentiates Aβ pathology in vivo, a model of AD, theAPP_(Swe)/PS1_(ΔE9) mouse (subsequently referred to as APP/PS1) was usedin which CD33 function was inactivated. APP/PS1 mice exhibit elevated Aβ(including Aβ42) production and develop amyloid plaques (Jankowsky etal., 2004). APP/PS1 mice lacking CD33 (APP/PS1/CD33^(−/−)) were born atMendelian ratios and exhibited no gross anatomical defects (data notshown). The cortex was isolated from four-month-old APP/PS1 andAPP/PS1/CD33−/− mice and their littermate controls and detergent-solublefractions were derived that were used for subsequent western blotting aswell as TBS-soluble and FA-soluble fractions (Wang et al., 2011) thatwere used for Aβ ELISA experiments. Using western blotting it was found,as expected, increased and similar levels of full-length APP and APPC-terminal fragments (APP-CTF) in both APP/PS1 and APP/PS1/CD33−/− miceas compared to controls (FIGS. 6A and 6B). BACE1 levels also did notdiffer across genotypes (data not shown).

Next it was investigated whether knockout of CD33 affects Aβ levels inthe APP/PS1 mice. As expected, it was found that Aβ40 and Aβ42 levels(measured by ELISA) in the TBS-soluble fraction were markedly increasedin APP/PS1 mice relative to WT and CD33^(−/−) mice (FIGS. 6C and 6D).Furthermore, the levels of Aβ40 and Aβ42 in the TBS-soluble fractionwere similar in the APP/PS1, APP/PS1/CD33^(+/−) and APP/PS1/CD33^(−/−)mice (FIGS. 6C and 6D). Remarkably, however, the levels of Aβ42, weresignificantly decreased in the FA-soluble fraction of APP/PS1/CD33^(−/−)mice relative to APP/PS1 mice (FIG. 6F, p<0.05 APP/PS1/CD33−/− versusAPP/PS1 mice, one-way Kruskal-Wallis ANOVA, Dunn's test). This effectwas not due to increased numbers of microglial cells or astrocytes inAPP/PS1/CD33^(−/−) mice (FIGS. 11A-11C) nor alterations in the levels ofneither inducible nitric oxide synthase, nor microglia-derivedcytokines, e.g. IL-1β and TNFα (data not shown). These data suggest thatCD33-deficient microglia possess increased Aβ42 uptake/clearancecapacity in vivo and that the CD33-mediated effect on microglialclearance of Aβ42 is cell-autonomous.

Finally, it was investigated whether CD33 deletion impacts the processof Aβ deposition in the APP/PS1 brain. Aβ deposition is obvious by 6months in APP/PS1 mice when both compact and diffuse Aβ-containingplaques can be observed in the forebrain (Jankowsky et al., 2004).Coronal sections from 6-7 month-old control, CD33^(−/−), APP/PS1 andAPP/PS1/CD33^(−/−) mice were stained with an antibody directed againstAβ (residues 1-5, 3D6) that recognizes both compact and diffuse Aβplaques (Reilly et al., 2003). The 3D6 antibody did not label anyplaques in control and CD33^(−/−) brains (data not shown). Analysis ofthe APP/PS1 brains revealed numerous Aβ plaques, both in the cortex andthe hippocampus (FIGS. 7A and 7C). Sections spanning the cortex or thehippocampus were subjected to quantification of amyloid plaque burden.Remarkably, the Aβ plaque burden was robustly decreased in theAPP/PS1/CD33^(−/−) brains relative to the APP/PS1 brains (FIGS. 7B and7D). The quantification revealed a 37.2% and 33.5% reduction of Aβplaque burden in the APP/PS1/CD33^(−/−) cortex and hippocampus,respectively (FIGS. 7E and 7F; n=9-11 animals/group, p<0.01 and p<0.05,APP/PS1/CD33^(−/−) versus APP/PS1 cortex and hippocampus, respectively,student's t-test). Therefore, CD33 promotes Aβ deposition and plaqueformation in vivo.

Discussion

Despite great strides towards the development of an effective therapy,no treatment is currently able to prevent, delay or stop AD progression.This is due in part to our incomplete understanding of AD pathogenesis.While important advances have been made towards a better understandingof FAD, comparatively little is known about LOAD. Great advances in DNAsequencing technologies coupled with the investigation of large humancohorts have provided an unprecedented analytic power and have led tothe identification of multiple risk factors for LOAD, thus opening newresearch avenues that might advance our understanding of thisdevastating disease.

The CD33 gene has been found to be associated with risk for LOAD(Bertram et al., 2008; Hollingworth et al., 2011; Naj et al., 2011).CD33 is a member of the Siglec family of lectins that bind sialic acidresidues leading to cis (same CD33-expressing cell) or trans (adjacentcells) interactions that regulate several aspects of innate immunity(Crocker et al., 2007; Paulson et al., 2012). To date, the role of CD33in the brain has remained unknown. To bridge this gap, and to understandhow CD33 misregulation contributes to LOAD pathogenesis, a comprehensiveanalysis of CD33 expression and mechanism of action was performed, usinga combination of human and mouse genetics, biochemistry, neuropathologyand in vitro modeling. Based on these findings, without wishing to bebound by theory, increased CD33 activity in microglial cells inhibits Aβclearance in LOAD; thus, CD33 inhibition represents a novel means forpreventing and treating AD.

CD33 Expression is Increased in AD.

It was found that the levels of CD33 protein are increased in AD. Thisincrease was paralleled by an increase in the number ofCD33-immunoreactive microglia. Moreover, the ratio CD33/Iba1 is alsoincreased in AD, suggesting that individual microglia display increasedCD33 levels in AD. Thus, AD cases are characterized by both an increasein the number of CD33-immunoreactive microglia and an increase in CD33levels in these CD33-positive cells. Moreover, the fact that the levelsof CD33 mRNA were also strongly increased in AD suggests a potentialupregulation of CD33 transcription in microglial cells; alternatively,the CD33 mRNA might exhibit increased stability in microglia in the ADbrain. It can be important to determine whether CD33 is part of abroader transcriptional program that operates in microglial cells in theaging brain and to identify the upstream activators of such a program.

To further explore the relationship between CD33 expression and AD, CD33expression was assessed in carriers of the minor (T) allele of the CD33SNP rs3865444, which protects against AD. It was found that the minor(T) allele is associated with reduced CD33 protein levels, both in theAD and control subjects (FIG. 1E). Interestingly, the levels of CD33mRNA were not reduced in the carriers of this allele. The rs3865444 SNPis located upstream of the 5′ untranslated region of the CD33 gene(Hollingworth et al., 2011; Naj et al., 2011). One possibility is thatthe rs3865444 SNP is in linkage disequilibrium with a functionalvariant(s) located in the coding region. This, in turn, could influencemRNA translation without affecting mRNA stability. Recently, increasedCD33 mRNA levels were independently shown to be associated with AD; CD33mRNA levels correlated with Iba1 mRNA levels and CD33 mRNA expressionnormalized to Iba1 expression correlated with disease status andClinical Dementia Rating scores (Karch et al., 2012). The increased CD33(mRNA and protein) levels in the AD brain and decreased CD33 proteinlevels in the carriers of the protective allele suggest that increasedCD33 expression plays a direct role in the etiology and/or pathogenesisof AD.

CD33 Is a Novel Modifier of Amyloid Beta Pathology.

Several genes associated with LOAD have been shown to be criticallyinvolved in the control of Aβ homeostasis. ApoE binds Aβ and influencesits oligomerization (Hashimoto et al., 2012). It was recently found thatlipidated apoE, in particular its pathogenic variant apoE4, increasesthe oligomerization of Aβ; as a consequence, APOE 84/84 AD brainsdisplayed higher levels of Aβ oligomers relative to APOE 83/83 brains(Hashimoto et al., 2012). The LOAD-associated gene Clusterin (CLU)encodes an extracellular chaperone apolipoprotein J (apoJ) thatinteracts with Aβ prefibrillar structures and inhibits Aβ aggregation(Yerbury et al., 2007). ApoJ also facilitates the transport ofplasma-derived Aβ across the blood brain barrier (BBB) and Aβ42clearance at the BBB in mice (Bell et al., 2007). Interestingly, apoJcooperates with apoE to suppress Aβ levels and deposition in vivo(DeMattos et al., 2004).

Besides the control of Aβ self-assembly, LOAD-associated genes are ableto control the trafficking of APP and thus influence the formation ofAβ. A genetic screen in yeast revealed that several LOAD genes(including PICALM and BIN1) were modifiers of Aβ toxicity, and suggestedthat these genes regulate the endocytic trafficking of APP (Treusch etal., 2011). Moreover, PICALM promotes APP internalization, endocytictrafficking and Aβ generation in neurons in vitro (Xiao et al., 2012).The ABCA7 LOAD risk gene also regulates APP trafficking, by stimulatingcholesterol efflux, which decreases the levels of APP at the plasmamembrane and Aβ generation (Chan et al., 2008). Therefore, targeting theintracellular trafficking of APP in neurons might represent a noveltherapeutic approach in AD.

CD33 was identified as a novel modifier of Aβ pathology in vivo. It wasobserved that an association existed between the protective minor (T)allele of the CD33 SNP rs3865444 and decreased FA-soluble Aβ42 levels inAD as well as a positive correlation between CD33 microglial expressionand FA-soluble Aβ42 levels and amyloid plaque burden in the AD cortex(FIG. 3). Moreover, CD33 levels directly impact microglial uptake ofAβ42 (FIGS. 4 and 5) and modulate the accumulation of FA-soluble Aβ42and Aβ plaque burden in APP/PS1 transgenic mice (FIGS. 6 and 7). Basedon these observations, without wishing to be bound by theory, theincreased activity of CD33 in microglial cells contributes to theetiology and/or pathogenesis of AD by preventing Aβ uptake and thuspotentiating its toxicity.

Taken together, the multiple lines of evidence implicating multiple LOADrisk genes in the control of Aβ production, clearance and depositionprovide additional support for the amyloid hypothesis of AD bydemonstrating that failure of multiple systems that ensure Aβhomeostasis is associated with an increased risk for developing AD.These findings also indicate immense therapeutic opportunities, sincethe identification of drug targets that are critically involved incontrol of Aβ pathogenicity by the novel LOAD risk genes might providenovel strategies targeting the earliest stages of cognitive decline,well before the occurrence of overt neurodegeneration.

CD33 Inhibits the Microglial Uptake of Amyloid Beta.

Based on the finding that CD33 microglial expression is elevated in ADbut decreased in carriers of the protective minor (T) allele of the CD33SNP rs3865444, it was hypothesized that the activity of CD33 inmicroglia promotes AD pathogenesis. To test this hypothesis and tounravel the molecular underpinnings of CD33 action in microglial cells,it was investigated whether CD33 is involved in the process of Aβclearance by microglia. Using an assay of microglial uptake andclearance optimized for Aβ42 (Jiang et al., 2008), it was found thatmouse primary microglial cells lacking CD33 expression exhibit anincreased uptake of Aβ42 relative to WT cells (FIGS. 4A-4D).Interestingly, CD33-deficient and WT cells degraded Aβ42 at a similarrate (FIG. 4E). In CD33-deficient cells, however, Aβ42 clearance wasaccelerated, due to the increased overall uptake. To further explore theinvolvement of CD33 in Aβ42 uptake by microglial cells, awell-characterized microglial cell line (BV2) was employed whicheffectively internalizes and degrades added Aβ42 (Mandrekar et al.,2009). It was found that microglia overexpressing WT CD33 were markedlyimpaired in their capacity to internalize Aβ42, but degraded theinternalized Aβ42 at a similar rate to cells transfected with an emptyplasmid or a plasmid encoding GFP (FIGS. 5E-5G). This finding was alsovalidated by the transfection with an ubiquitylation-defective CD33mutant (CD33^(K7R)), which exhibits enhanced cell surface expression ofCD33 (FIG. 5C and (Walter et al., 2008)) and further exacerbates theinhibition of Aβ42 uptake (FIGS. 5E and 5F). Thus, CD33 directlymodulates uptake, but not degradation, of Aβ42 by microglial cells.

CD33 and the related Siglecs perform their biological functions byinteracting with sialic acids, which are attached to the outer membraneof cells, and can mediate cis- or trans-cellular interactions (Crockeret al., 2007). To explore the requirement of sialic acid binding in theprocess of CD33-mediated microglial uptake of Aβ42, microglial BV2 cellswere transfected with a CD33 mutant in which the sialic acid-bindingV-type immunoglobulin-like domain was removed. The mutant CD33^(ΔV-Ig)protein is present at the plasma membrane in BV2 cells, and is expressedat levels similar to CD33^(WT) (FIGS. 5B and 5D and (Perez-Oliva et al.,2011)). Cells expressing the CD33^(ΔV-Ig) protein were no longerimpaired in their capacity to uptake Aβ42, indicating that sialic acidbinding is necessary for the ability of CD33 to inhibit Aβ42 uptake.Collectively, these experiments indicate that CD33 directly modulatesmicroglial uptake of Aβ42, via interaction with sialic acids.

Microglial cells have been suggested to play critical roles as mediatorsof Aβ clearance in the brain. A previous study (Grathwohl et al., 2009)challenged this view by showing that ablation of microglial cells (usingdrug-induced microglial toxicity) did not alleviate Aβ pathology in twoAD mouse models, within a 4 week time-window following the ablation.These results await confirmation in a setting of prolonged microglialablation (several months to years). It also remains to be determinedwhether an acute removal of microglial cells has a specific effect onthe dynamics of monomeric and oligomeric as opposed to fibrillar Aβ.

Mounting evidence implicates genes associated with risk for LOAD in theprocess of microglial clearance of Aβ42. For example, apoE promotes theclearance of Aβ in the brain, partly through its capacity to enhance Aβuptake and degradation by microglia (Jiang et al., 2008; Lee et al.,2012). Interestingly, another LOAD risk gene, ApoJ/Clusterin (CLU)cooperates with APOE to suppress Aβ levels and deposition in PDAPPtransgenic mice (DeMattos et al., 2004). A rare variant of the TREM2gene has been recently shown to confer increased risk for LOAD(Guerreiro et al., 2013; Jonsson et al., 2013). TREM2 is an innateimmune receptor, similar to CD33, and is expressed in a subset ofmyeloid cells including microglia (Klesney-Tait et al., 2006).Remarkably, TREM2 is upregulated in amyloid plaque-associated microgliain aging APP23 transgenic mice (Frank et al., 2008) and in CRND8transgenic mice (expressing mutant KM670/671NL and V717F APP) (Guerreiroet al., 2013) and promotes the phagocytic clearance of amyloid proteins(Melchior et al., 2010). Interestingly, TREM2 interacts with its ligandTREM2-L, expressed by apoptotic neurons, and mediates removal of dyingneuronal cells by microglia (Hsieh et al., 2009).

The results described herein suggest that CD33 represents a novelregulator of microglial clearance of Aβ and a novel target for thetreatment and prevention of AD. It is interesting to note that therapiestargeting CD33 have already been developed in acute myeloid leukemia(AML), due to its high membrane expression in myeloid cells (Jandus etal., 2011; O'Reilly and Paulson, 2009). Naked humanized anti-CD33 andcalicheamicin-conjugated humanized murine anti-CD33 antibodies have beendeveloped and tested in several phase III clinical trials of AML, withmixed results (Jandus et al., 2011; Jurcic, 2012; Ricart, 2011). Thissuggests that the development of a CD33 antibody that is able to crossthe BBB is feasible in principle. A chimeric antibody in which the CD33antibody is fused to a monoclonal antibody against the human insulinreceptor could facilitate the receptor-mediated passage of the chimeraacross the BBB (O'Reilly and Paulson, 2009). An alternative approach isthe development of small compounds, e.g. sialic acid-based antagoniststhat target CD33 specifically and inhibit its function. Furthermore, abetter understanding of CD33 action in microglial cells should lead tothe identification of critical cellular targets that link the activityof CD33 in microglia to the process of Aβ recognition and uptake, andcan lead to the development of novel therapeutics for the prevention andtreatment of AD.

Experimental Procedures

SNP Genotyping.

Genomic DNA was extracted from AD and control frozen brain tissue usingthe QIAamp DNA Mini Kit (Qiagen). DNA Samples were genotyped for SNPsusing the TaqMan SNP genotyping assays (Shen et al., 2009). Thegenotyping was performed using a custom-designed TaqMan® SNP genotypingassay for the rs3865444 SNP (Life Technologies) on a CFX384 Real-TimePCR System (BioRad) in accordance with the supplier's recommendations.To determine the APOE genotype (alleles: ε2, ε3, ε4), DNA samples weregenotyped at two SNPs, rs429358 and rs7412, using a pre-designed TaqmanSNP genotyping assay (catalogue no. 4351379) from Life Technologies. Thereactions were carried out in 384-well microtiter plates (BioRad) in atotal reaction volume of 5 ul, containing 1 μl genomic DNA (5 ng/μl),2.5 μl of 2× Gene expression master mix (Life Technologies), 0.063 ul of80× Taqman probes, and 1 ul water. Thermal cycling was performed with 40cycles of 92° for 15 seconds and 60° for 30 seconds. Data was analyzedusing the CFX Manager Software and the allelic discrimination tool.

RNA Extraction and Real Time-PCR.

RNA was extracted from brain tissue with Trizol (Life Technologies)following manufacturer's instructions. The extracted mRNA was dissolvedin water and cleaned using the RNAeasy Mini Kit (Qiagen) according tothe manufacturer's protocol. Extracted mRNA (2 μg) wasreverse-transcribed using the SuperScript III first strand synthesissystem (Life Technologies). Gene expression was assessed by performingTaqman real-time PCR assays. Probes targeting the CD33 gene were labeledwith FAM (Hs01076280_g1 specific to exons 3-4 and Hs01076281_m1 specificto exons 4-5, Life Technologies). The housekeeping genes GAPDH andβ-Actin were used as controls and were labeled with a VIC/MGB probe(Life Technologies, 4326315E for β-Actin and 4326317E for GAPDH). 1:10diluted cDNAs were mixed with the probes and 2× Gene expression mastermix (Life Technologies) and amplified using a CFX384 Real-Time PCRSystem (BioRad). Results were analyzed by the comparative CT method.Average CT values for each sample were normalized to the average CTvalues of the housekeeping genes. GAPDH expression was highly correlatedwith β-Actin expression.

Immunohistochemistry and Stereology.

For immunohistochemistry, mice were deeply anesthetized with isoflurane,then perfused with 0.9% sodium chloride and ice-cold 4% paraformaldehyde(PFA). Subsequently, brains were removed from the skull and post-fixedovernight in 4% PFA. The left hemisphere was dehydrated with ethanol andembedded in paraffin, whereas the right hemisphere was cryoprotected byincubation in 15% and 30% sucrose solutions and embedded in OCT.

Sections (8-μm thick) were deparaffinized and incubated with 3% H₂O₂ toquench endogenous peroxidases for DAB staining Antigen retrieval wasperformed using the Diva Decloaker (Biocare Medical) or citrate buffer(0.01M, pH 6.0, 0.05% Tween-20) in a microwave oven (95° C., 20minutes). Sections were subsequently blocked using 2% BSA, 0.1% TritonX-100 in phosphate buffered saline (PBS), or alternatively with Antibodydiluent (Cell signaling). Primary antibodies were directed against humanCD33 (mouse monoclonal, 1:100, clone PWS44, Vector Laboratories) (Hoyeret al., 2008; Rollins-Raval and Roth, 2012); MAP2 (rabbit polyclonal,1:500, Millipore); Iba1 (rabbit polyclonal, 1:500, Wako); human GFAP(rabbit polyclonal, 1:1,000, Sigma); mouse GFAP (mouse monoclonal,1:500, clone GA5, Millipore); human von Willebrand factor (rabbitpolyclonal, 1:500, Millipore); human myelin basic protein (rabbitpolyclonal, 1:300, Millipore). To detect amyloid plaques, sections werelabeled with 1% Thioflavin S in water for 8 minutes at room temperature,then incubated in 80% ethanol for 2 minutes and mounted. For theimmunostaining with the antibodies targeting human Aβ (mouse monoclonal,clone 10D5 [1:50] or clone 3D6 [1:2000], Elan Pharmaceuticals), antigenretrieval was performed using citrate buffer, in a microwave oven (95°C., 20 minutes) followed by incubation in 90% formic acid for 5 minutes.Biotinylated secondary antibodies were from Vector Laboratories.

For immunofluorescence experiments, Alexa-488/564/647-coupled secondaryantibodies and Alexa 568-coupled streptavidin were acquired from LifeTechnologies. Sections were mounted with aqueous mounting mediumcontaining DAPI and anti-fading reagent (Life Technologies).

For stereology-based quantitative studies, primary antibodies weredetected with DAB, using different Vectastain ABC kits (VectorLaboratories) according to the provider's instructions. Sections weredehydrated with increasing concentrations of ethanol, cleared withxylene, and cover-slipped with Cytoseal-XYL xylene-based mounting medium(Richard-Allan Scientific).

An unbiased stereology-based quantification method was used to determinethe number of CD33-positive microglia and neurons on singleimmunostained sections from human frontal cortex (Brodmann areas 8, 9)(Serrano-Pozo et al., 2011). Briefly, sections were placed on themotorized stage of an upright Olympus BX51 microscope that is equippedwith a DP70 video-camera and controlled by a computer with the imageanalysis software CAST (Olympus, Tokyo, Japan). The region of interest(full-width) cortex was outlined under the 4× objective andCD33-positive cells were counted under the 40× objective using a 1%meander sampling and a 10% counting frame (3565.2 μm²).

Assessment of Aβ plaque burden was performed in an upright Leica DMRBmicroscope (Leica, Germany) equipped with a motorized stage and a CCDcamera and coupled with the software BIOQUANT NOVA PRIME, version6.90.10 (MBSR). Aβ plaque burden was measured as the percentage of totalsurface stained by the anti-Aβ antibody (clone 10D5) in a full-widthstrip of cortex (approximately 1-cm long) using the optical thresholdapplication of the software (Serrano-Pozo et al., 2011).

Assessment of Aβ Plaque Burden in Mice.

Coronal sections stained with the anti-Aβ antibody 3D6 (1:2000, ElanPharmaceuticals) were imaged using an upright Olympus BX51 microscope.Six coronal sections spanning the cortex and four coronal sectionsspanning the hippocampus (at different depths on the rostro-caudal axis)were imaged for each animal. The amyloid plaque burden (area occupied byall plaques divided by the total area) was estimated in the cortex orhippocampus for each section using ImageJ software. Values from eachsection were then averaged to derive a mean plaque burden for eachanimal. 9-11 male mice were analyzed in each group (age 6-7 months).

ELISA.

For ELISA, mice were deeply anesthetized with isoflurane, then perfusedwith 0.9% sodium chloride. The brains were extracted and cortices andhippocampi were dissected. To assess Aβ levels, mouse cortices or humanfrozen brain tissue (frontal cortex) were homogenized in 5 volumes ofTBS containing 5 mM EDTA, phosphatase inhibitor (ThermoFisher),EDTA-free protease inhibitor cocktail (Roche) and 2 mM1,10-phenantroline (Sigma), using a Polytron benchtop lab homogenizer(Wheaton) at 4° C. The homogenate was centrifuged at 100,000 g for 1hour at 4° C. using an Optima TL ultracentrifuge and a TLA 120.2 rotor(Beckman Coulter). Supernatants were collected and used to measureTBS-soluble Aβ. The resulting pellet was homogenized in 70% formic acid(the volume of used formic acid was equal to the volume of TBShomogenate used for centrifugation). Samples were centrifuged at 100,000g for 1 hour at 4° C. and supernatants were collected. Formicacid-containing supernatants were neutralized with 1M Tris-base, pH 11(1:20 v:v) and samples were used to measure formic acid-soluble Aβ. Aβ40and Aβ42 ELISAs were performed using Aβ ELISA kits from Wako.

Western Blot Analysis.

Brain tissue was homogenized in 5 volumes of TBS containing 5 mM EDTA,phosphatase inhibitor (ThermoFisher), EDTA-free protease inhibitorcocktail (Roche) and 2 mM 1,10-phenantroline (Sigma). The homogenate wasmixed with an equal volume of 2× radio-immunoprecipitation assay (RIPA)buffer (Millipore) supplemented with all the above-described inhibitors.Samples were incubated on a rotating wheel for 30 minutes at 4° C. andcentrifuged at 12,000 g for 15 minutes at 4° C. The supernatant wascollected and used for western blot analysis. Lysates were assessed forprotein concentration using the BCA kit (Pierce). Samples were boiled insample buffer containing lithium dodecyl sulfate and β-mercaptoethanolas reducing agent (Life Technologies), and resolved on 4%-12% Bis-Trispolyacrylamide precast gels (NuPAGE system, Life Technologies). Gelswere transferred onto PVDF membranes (BioRad) using wet transfer system(BioRad). The primary antibodies were directed against: human CD33(mouse monoclonal, 1:100, clone PWS44, Vector Laboratories); Iba1(rabbit polyclonal, 1:500, Wako); GAPDH (mouse monoclonal, 1:10,000,Millipore); β-Actin (mouse monoclonal, 1:5,000, Sigma), APP (rabbitpolyclonal, 1:2,000, clone C7 targeting the amino acid residues 732-751in APP, custom-designed by Open Biosystems) (Podlisny et al., 1991) andmouse CD33 (mCD33, rabbit polyclonal, 1:200, custom-designed by OpenBiosystems). Densitometric analyses were performed using Quantity Onesoftware (BioRad). Band density values were normalized to GAPDH or13-Actin levels.

Generation of an Anti-Mouse CD33 Antibody.

mCD33 is a rabbit polyclonal antibody that was generated by OpenBiosystems. It was raised against a mouse CD33 epitope that is absent inother mouse CD33-related Siglecs and corresponds to amino acid residues18-32 (DLEFQLVAPESVTVE). The antibody was characterized by western blotanalysis and immunocytochemistry (FIGS. 4 and 10).

Primary Microglia Isolation.

Microglial cells were prepared from WT or CD33^(−/−) brains at postnatalday 1 as previously described (Choi et al., 2008; Gorlovoy et al.,2009). Briefly, meninges and leptomeningeal blood vessels were removedfrom the cortex. Cells were dissociated by trituration and cultured inDMEM containing 10% heat-inactivated fetal bovine serum, 2 mML-glutamine and 1% penicillin/streptomycin (Life Technologies) for 14-21days in poly-D-lysine-coated 75 cm2 flasks (Biocat) to form a confluentglial monolayer. Half of the medium was replaced with fresh cell culturemedium every three days. To collect microglial cells, the cultures wereshaken on a rotary shaker (placed in a cell culture incubator, 37° C.and 5% CO2) at 250 rpm for 3 hours. The detached microglial cells werecollected by centrifugation and the enriched microglial cell suspensionwas plated onto poly-D-lysine-coated 6-well plates (Biocat) or onto24-well plates containing poly-D-lysine-coated glass coverslips. Afterthe cells attached, the medium was replaced with fresh cell culturemedium. The purity of the isolated microglia was determined byimmunostaining with antibodies directed against Iba1. In average, 93% ofcultured cells were immunostained with Iba1.

Cell Culture and Transfection.

Two pcDNA3.1 plasmids encoding human wild-type CD33 (CD33WT) and mutantCD33K283/288/309/312/313/315/352R (CD33K7R) were generously provided byRoland B. Walter and were previously described (Walter et al., 2008a).pCMV6-XL5 plasmid encoding CD33 that lacks the sialic acid-bindingV-type immunoglobulin-like domain (CD33ΔV-Ig) was acquired from OriGene.CD33ΔV-Ig cDNA was amplified and subcloned into a pcDNA3.1 vector (LifeTechnologies). All constructs were verified by sequencing. BV2microglial cell line was kindly provided by Linda Van Eldik (Bachstetteret al., 2011) and was maintained in DMEM containing 5% heat-inactivatedfetal bovine serum, 2 mM L-Glutamine and 1% penicillin/streptomycin(Life Technologies). Cells were transiently transfected with CD33plasmids using Lipofectamine-Plus (Life Technologies) according to themanufacturer's instructions.

Aβ Uptake and Degradation Assays.

Primary mouse microglia or BV2 cells transfected with CD33 plasmids weretreated with 2 μg/ml Aβ42 (AnaSpec) in serum-free DMEM medium for 3hours. Cells were washed with DMEM and maintained for additional 3 hoursin serum- and Aβ42-free DMEM. Afterwards, cells were extensively washedwith PBS and were lysed in cell lysis buffer (50 mM Tris-HCl, pH 7.4,150 mM NaCl, 5 mM EDTA, 1% SDS) supplemented with EDTA-free proteaseinhibitors (Roche) and 2 mM 1,10 phenantroline (Sigma). Lysates werecentrifuged at 12,000 g at 4° C. for 15 minutes. Supernatants werecollected and further used for ELISAs. Aβ42 levels were measured usingAβ42 ELISA kit (Wako) and normalized to total protein concentration thatwas assessed by BCA method (Pierce).

For immunofluorescence experiments, primary mouse microglial or BV2cells were incubated with 2 μg/ml Alexa 555-labeled Aβ42 (AnaSpec) inserum-free DMEM for 3 hours, washed extensively with PBS and fixed with4% PFA for 20 minutes. Cells were washed with PBS, blocked with 4% BSAin PBS and incubated with the primary antibodies targeting: mouse CD33(rabbit polyclonal, 1:200, custom-designed by Open Biosystems), humanCD33 (mouse monoclonal, 1:200, clone PWS44, Vector Laboratories) andIba1 (rabbit polyclonal, 1:500, Wako, or goat polyclonal, 1:500, Abcam).Alexa 488/647 conjugated secondary antibodies were purchased from LifeTechnologies. Coverslips were mounted with aqueous mounting mediumcontaining DAPI and anti-fading reagent (Life Technologies).

To quantify intracellular Alexa555-labeled Aβ42, pictures displaying theintracellular Aβ42 in transfected BV2 or mouse primary microglial cellswere used. The area corresponding to intracellular Aβ42 was carefullydelineated and the average intensity of the signal was determined withinthis area. To correct for differences in background intensity, an areadevoid of Aβ42 was used and the average intensity in this area wassubtracted from the average intensity of Aβ42 signal. Thequantifications were performed using the ImageJ software and the valuesare represented in arbitrary units. At least 30 cells were analyzed percondition.

Brain Specimens.

Formalin-fixed, paraffin-embedded 8 micron-thick sections, as well asfrozen tissue specimens from the frontal cortex of 25 patients with ADand 15 age-matched non-demented control subjects were obtained from theMassachusetts Alzheimer's Disease Research Center Brain Bank. All thestudy subjects or their next of kin gave written informed consent forthe brain donation, and the Massachusetts General Hospital InstitutionalReview Board approved the study protocol. The demographiccharacteristics of both groups are shown in Table 1. All patients withAD fulfilled the National Institute of Neurological and CommunicativeDisorders and Stroke-Alzheimer's Disease and Related DisordersAssociations criteria for probable AD and the National Institute onAging-Reagan Institute criteria for high likelihood of AD.

Animals.

APP_(Swe)/PS1_(ΔE9) transgenic mice (referred to as APP/PS1) (Jankowskyet al., 2004) and constitutive CD33 knockout mice (Brinkman-Van derLinden et al., 2003) were obtained from The Jackson Laboratory(catalogue no. 005864 and 006942, respectively). Both mouse strains areon the C57Bl/6 background. All mice were housed under standardconditions with free access to food and water. All animal experimentswere performed in accordance with national guidelines (NationalInstitutes of Health) and approved by Massachusetts General Hospital andMcLaughlin Institute Institutional Animal Care and Use Committees.

Statistical Analysis.

A general linear regression model adjusting for appropriate covariateswas used to test for allelic association between the rs3865444 SNP andquantitative traits, as implemented in PLINK v1.07(http://pngu.mgh.harvard.edu/purcell/plink/) (Purcell et al., 2007). Toidentify covariates that maximize the regression model's predictiveability and predict the quantitative traits for the human samples, astepwise regression procedure was performed using age, gender, diseasestatus, post-mortem interval, and presence of APOE ε4 allele. Thestepwise regression analysis was performed using the R (v2.10.0)software package (R Development Core Team, 2009). Statistics andcorrelations of different quantitative traits were performed using theGraphPad Prism software, version 5.0 (GraphPad Inc., La Jolla, Calif.).The normality of quantitative trait data sets was tested with theD'Agostino-Pearson omnibus test. Student's t-test and Pearson'scorrelation test were performed for normally-distributed data sets, andMann-Whitney U and Spearman's correlation tests otherwise. Multiplegroup analyses were performed by one-way analysis of variance (ANOVA)followed by Tukey's post-hoc test or by one-way Kruskal-Wallis ANOVAfollowed by Dunn's post-hoc test.

REFERENCES FOR EXAMPLE 1

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All patents and other publications identified in the specification andexamples are expressly incorporated herein by reference for allpurposes. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedcan be further modified to incorporate features shown in any of theother embodiments disclosed herein.

1. A method of treating a neuro-inflammation disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of an agent that inhibits or reduces the expression or activity of CD33 protein. 2.-4. (canceled)
 5. The method of method of claim 1, wherein the agent is a nucleic acid and the method comprises administering a vector encoding/expressing the agent to the subject.
 6. The method of claim 5, wherein the vector is a viral vector
 7. The method of claim 6, wherein the viral vector is an adeno-associated virus (AAV) vector.
 8. The method of claim 1, wherein the agent is a small molecule selected from the group consisting of sialic acid analogues and derivatives.
 9. The method of claim 1, wherein the agent is a monoclonal antibody.
 10. The method of claim 1, wherein the agent is a humanized antibody.
 11. The method of claim 1, wherein the agent is an anti-CD33 antibody or an antigen binding fragment thereof.
 12. (canceled)
 13. A method of treating a neuro-inflammation disorder in a subject, the method comprising administering to a subject in need thereof a nucleic acid encoding a CD33 protein, wherein the CD33 protein lacks sialic acid binding domain.
 14. The method of claim 13, wherein the CD33 protein comprises an amino acid sequence SEQ ID NO:
 8. 15. The method of claim 13, wherein the nucleic acid is a vector.
 16. The method of claim 15, wherein the vector is a viral vector
 17. The method of claim 16, wherein the viral vector is an AAV vector.
 18. The method of claim 13, wherein the nucleic acid is modified RNA.
 19. (canceled)
 20. The method of claim 1, wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's Disease, Amyotrophic Lateral Sclerosis (ALS, also termed Lou Gehrig's disease) and Multiple Sclerosis (MS).
 21. The method of claim 20, wherein the neurodegenerative disorder is Alzheimer's Disease. 22.-23. (canceled)
 24. The method of claim 1, wherein the agent decreases beta amyloid accumulation in the brain of the subject.
 25. The method of claim 24, wherein the beta amyloid is Aβ-42.
 26. A method of decreasing beta amyloid accumulation in the brain of a subject, the method comprising administering to a subject in need thereof an effective amount of an agent that inhibits or reduces the expression or activity of CD33 protein. 27.-37. (canceled)
 38. A method of decreasing beta amyloid accumulation in the brain of a subject, the method comprising administering to a subject in need thereof a vector expressing administering to a subject in need thereof a nucleic acid encoding a CD33 protein, wherein the CD33 protein lacks sialic acid binding domain. 39.-52. (canceled) 